A 2:1:1 mixture of: trisodium N(1')-N(2):N(1''')-N(2'')-η-6-[2-amino-4-(or 6)-hydroxy-(or 4-amino-2-hydroxy)phenylazo]-6''-(1-carbaniloyl-2-hydroxyprop-1-enylazo)-5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'-azobenzene-1,2'-diolato-O(1),O(2'))-chromate; trisodium N(1')-N(2):N(1''')-N(2'')-η-6,6''-bis(1-carbaniloyl-2-hydroxyprop-1-enylazo)-5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'azobenzene-1,2'-diolato-O(1),O(2'))-chromate; trisodium N(1')-N(2):N(1''')-N(2'')-η-6,6''-bis[2-amino-4-(or 6)-hydroxy-(or 4-amino-2-hydroxy)phenylazo]5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'azobenzene-1,2'-diolato-O(1),O(2'))-chromate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29. Aug. to 29. Oct. 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Remarks:

Dose-range finding

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 30th May to 23rd June, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to other study

Remarks:

main study

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 0, 150, 400 and 1000 mg/kg/day and the effects on pregnancy and embryo-foetal development in the rat are investigated.

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 (200-225 g)
♂ 11 weeks old (at least 320 g)
- Housing:
During the pre-pairing period and after mating: no more than 5 of one sex to a cage, in polysulfone cages measuring 59.5×38×20 cm. Nesting material was provided inside suitable bedding bags and changed at least 2 times a week.
During the mating period: on the basis of 1 male to 1 female in clear polysulfone cages measuring 4 2.5x26.6x18.5 cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily.
- Diet: a commercially available laboratory rodent diet was offered ad libitum throughout the study.
- Water: ad libitum to each cage via water bottles
- Acclimation period: 13 days

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

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

softened by reverse osmosis

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The preparations were made daily (concentrations of 15, 40 and 100 mg/mL) and the concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
The test item was administered at a dose volume of 10 mL/kg body weight.

Details on mating procedure:

The females were paired with male rats. Females were paired one to one in the home cage of the male and left overnight. Vaginal smears were taken daily in themorning from the day after pairing until a positive identification of mating was made. The day of mating, as judged by the presence of spermin the vaginal smear or by the presence of a copulationplug, was considered as Day 0 of ge station (or Day 0 post coitum). Full mating records were maintained.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 days

Frequency of treatment:

once a day

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

low dose

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

medium dose

Dose / conc.:

1 000 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:

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 5 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.

Parental animals: Observations and examinations:

Full records were maintained for all measurements and observations.

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.

CLINICAL SIGNS
All clinical signs were recorded for individual animals. Each animal was observed at least once daily and any clinical signs recorded starting from allocation until sacrifice.

BODY WEIGHT
All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.

Postmortem examinations (parental animals):

EUTHANASIA
The animals were killed by exsanguination under isoflurane anaesthesia on Day 20 post coitum and necropsied. All foetuses were sacrificed by intraperitoneal injection of Sodium Thiopental followed by hypothermia.

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.
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 termwithout 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.

EXAMINATION OF FOETUSES
All viable foetuses were euthanised and externally examined, after which the carcasses were discarded.
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:

Standard deviations were calculated as appropriate. For the continuous variables (e.g. body weight, body weight gain), 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. The non-parametric Kruskal-Wallis analysis of variance (non-continuous variables) was used for the other parameters (e.g. uterus weight, number of implantation sites, litter data, sex ratios). Intergroup differences between the control and treated groups were assessed by the non-parametric version of theWilliams test. The criterion for statistical significance was p < 0.05.
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.
The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed throughout the study in control and treated females.

Mortality:

no mortality observed

Description (incidence):

No animals died during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No differences in bodyweight were noted throughout the study between control and treated groups.
A slight decrease in body weight gain was noted in all treated groups, compared to the control on Day 15 post coitum (-21 % to -33 %).
Terminal body weight, uterus weight and absolute weight gain of treated females were comparable to the control group.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

FATE OF FEMALES
All females were found pregnant at necropsy with the exception of one female receiving 400 mg/kg/day.
One female of Group 4 (1000 mg/kg/day) showed unilateral implantation in the right uterine horn and was not pregnant in the left one.
The number of females with live foetuses on Day 20 post coitum was: 6 in the control group, 6 in the low dose (150 mg/kg/day), 5 in the mid-dose (400 mg/kg/day) and 6 in the high dose (1000 mg/kg/day) groups.

Reproductive performance:

no effects observed

Description (incidence and severity):

LITTER DATA AND SEX RATIOS
A trend towards an increase in early uterine deaths (conceptus which, being implanted in the uterus, subsequently died or was reabsorbed) was noted in females receiving the dose levels ≥ 400 mg/kg/day. As a consequence, post-implantation and total implantation losses were increased, but they were not significant, at statistical analysis, even if with dose relationship. However, these alterations cannot be conclusively attributed to treatment since two out of 5 or 6 pregnant females were involved, both at 400 and 1000 mg/kg/day, respectively.
No differences were noted in sex ratios of the treated groups, when compared to the control.

EXTERNAL EXAMINATION OF FOETUSES
One small foetus (< 2.7 g) was detected in one control female.
No other abnormalities were detected at the external examination of foetuses.

Remarks on result:

other: the high dose level for the subsequent main study could be up to 1000 mg/kg/day

Conclusions:

The high dose level for the subsequent Reproduction/Developmental Toxicity Screening Test could be up to 1000 mg/kg/day, based on the results obtained.

Executive summary:

The effects of the test item were investigated after oral administration in female rats during pregnancy and on embryo-foetal development. All animals were administered during the gestation period, starting from Day 6 through Day 19 post coitum at the dose volume of 10 mL/kg. Each treatment group comprised 6 mated female rats and received the test item orally at the dose levels of 0, 150, 400 and 1000 mg/kg/day. The vehicle was softened water (by reverse osmosis).

The following investigations were performed: mortality check, clinical signs, body weight and macroscopic observation of the females. On Day 20 post coitum at necropsy, gravid uterus, absolute weight gain, corpora lutea, number of implantation sites, number, sex and weight of all live foetuses were determined. Gross evaluation of the placenta was also performed. All live foetuses were examined externally.

No mortality occurred during the study.

One female of the mid-dose group was found not pregnant at necropsy and one female of the high-dose showed unilateral implantation.

The number of females with live foetuses on Day 20 post coitum was 6 in the control group, 6 in the low-dose group, 5 in the mid-dose group and 6 in the high-dose group.

No signs of toxicity were observed during the dosing period.

No relevant differences in body weight were noted between control and treated groups. Body weight gain was slightly decreased on Day 15 in all treated groups, when compared to the controls.

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

At the dose levels ≥ 400 mg/kg/day some alterations in litter data, such as an increase in early uterine deaths or in post implantation and total implantation losses were recorded, when compared to the control group. However, these variations cannot be conclusively attributed to treatment since the total number of females affected was the same in both treated groups (2 out of 5 or 6).

No differences were noted in sex ratios of the treated groups, when compared to the controls.

Animals killed at termination did not show relevant macroscopic changes that could be considered treatment-related.

No abnormalities were detected at the external examination of treated foetuses.

On the basis of the above results, it can be concluded that the high dose level for the subsequent toxicological studies could be up to 1000 mg/kg/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

A 2:1:1 mixture of: trisodium N(1')-N(2):N(1''')-N(2'')-η-6-[2-amino-4-(or 6)-hydroxy-(or 4-amino-2-hydroxy)phenylazo]-6''-(1-carbaniloyl-2-hydroxyprop-1-enylazo)-5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'-azobenzene-1,2'-diolato-O(1),O(2'))-chromate; trisodium N(1')-N(2):N(1''')-N(2'')-η-6,6''-bis(1-carbaniloyl-2-hydroxyprop-1-enylazo)-5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'azobenzene-1,2'-diolato-O(1),O(2'))-chromate; trisodium N(1')-N(2):N(1''')-N(2'')-η-6,6''-bis[2-amino-4-(or 6)-hydroxy-(or 4-amino-2-hydroxy)phenylazo]5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'azobenzene-1,2'-diolato-O(1),O(2'))-chromate

EC Number:

402-850-1

EC Name:

A 2:1:1 mixture of: trisodium N(1')-N(2):N(1''')-N(2'')-η-6-[2-amino-4-(or 6)-hydroxy-(or 4-amino-2-hydroxy)phenylazo]-6''-(1-carbaniloyl-2-hydroxyprop-1-enylazo)-5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'-azobenzene-1,2'-diolato-O(1),O(2'))-chromate; trisodium N(1')-N(2):N(1''')-N(2'')-η-6,6''-bis(1-carbaniloyl-2-hydroxyprop-1-enylazo)-5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'azobenzene-1,2'-diolato-O(1),O(2'))-chromate; trisodium N(1')-N(2):N(1''')-N(2'')-η-6,6''-bis[2-amino-4-(or 6)-hydroxy-(or 4-amino-2-hydroxy)phenylazo]5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'azobenzene-1,2'-diolato-O(1),O(2'))-chromate

Molecular formula:

not applicable for UVCB substance

IUPAC Name:

trichromium(3+) nonasodium 6-[2-(2-amino-4-hydroxyphenyl)diazen-1-yl]-2-[2-(2-oxido-5-sulfamoylphenyl)diazen-1-yl]-3-sulfonatonaphthalen-1-olate 6-[2-(2-amino-6-hydroxyphenyl)diazen-1-yl]-2-[2-(2-oxido-5-sulfamoylphenyl)diazen-1-yl]-3-sulfonatonaphthalen-1-olate 6-[2-(4-amino-2-hydroxyphenyl)diazen-1-yl]-2-[2-(2-oxido-5-sulfamoylphenyl)diazen-1-yl]-3-sulfonatonaphthalen-1-olate tris(6-{2-[(1Z)-2-hydroxy-1-(phenylcarbamoyl)prop-1-en-1-yl]diazen-1-yl}-2-[2-(2-oxido-5-sulfamoylphenyl)diazen-1-yl]-3-sulfonatonaphthalen-1-olate)

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat is the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.

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
- Age at study initiation: (P) x wks; (F1) x wks
- Weight at study initiation: males: 215 to 235 g; femalesL 190 to 205 g
- Housing from arrival to pairing: up to 5 animals of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese); nesting material was provided inside suitable bedding bags and changed at least twice a week
- Housing during mating: one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a 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
- Housing after mating: males were re-caged as they were before mating; females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (remainder of study) (measuring 42.5×26.6×18.5 cm); nesting material was provided inside suitable bedding bags; in addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary and changed at least 2 times a week
- Diet: commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy); offered ad libitum
- Water: ad libitum to each cage via water bottles
- Acclimation period: 26 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 2 °C
- Relative humidity: 55 % ± 15 %
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

softened by reverse osmosis

Details on exposure:

- Dose administered: 10 mL/kg bw
PREPARATION OF TEST ITEM
The formulation was prepared daily (concentrations of 10, 30 and 100 mg/mL), unless specified otherwise.
Concentrations were calculated and expressed in terms of test item as supplied.

Details on mating procedure:

- M/F ratio per cage: 1 male and 1 female per mating cage (monogamous)
- Length of cohabitation: until positive identification occurred or 14 days had elapsed
- Proof of pregnancy: a vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually during gestation, and then remained in own cage during birth and lactation periods until the end of the study

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed in a separate study (see IUCLID section 8) in order to validate the analytical method and the preparation procedure. Final results for all levels were within the acceptability limits stated in RTC SOPs for concentration (90-110 %).
The stability of the preparations was also verified in the same study in the range from 5 to 100 mg/mL after 28 hours at room temperature and 10 days at +5 °C±3 °C.
Samples of the preparations prepared on Week 1 and last week of treatment (when all animals were present) were analysed to check the concentration. Resultsof the analyses were within the acceptability limits stated in RTC SOPs for concentration of solutions (90-110 %).
Chemical analysis was carried out by the Analytical Chemistry Department using a spectrophotometric detection (Multiskan spectrum (Thermo Electron Corporation). The software used for this activity was SkanIt software 2.4.2.55 (Thermo Electron Corporation).

Duration of treatment / exposure:

Males: 30 days
Females: minimum 51 days

Frequency of treatment:

once a day, 7 days a week

Details on study schedule:

- Pre-mating phase: two weeks
- Mating phase: 2 to 14 days
- Post-mating phase (males only): 15 days; until sacrifice on day 31
- Gestation phase: approximately 22 days post coitum (parturition check was performed daily from day 20 to day 25 post coitum)
- Lactation phase: 14 days post partum

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

negative control group; received vehicle only

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

low-dose group

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

mid-dose group

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

high-dose group

No. of animals per sex per dose:

10 males and 10 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on a preliminary dose range finding study
- Fasting period before blood sampling for clinical biochemistry: overnight

Positive control:

no

Examinations

Parental animals: Observations and examinations:

MORTALITY
- Daily, early in each working day in the morning and 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

CLINICAL SIGNS
- Once before commencement of treatment and at least once daily during the study; at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions

BODY WEIGHT
- Males were weighed weekly from allocation to termination; females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1, 4, 7, 13 post partum and just before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE
- The weight of food consumed by each cage of males and females was recorded weekly (whenever possible) during the pre-mating period starting from allocation; individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum.

CLINICAL PATHOLOGY INVESTIGATIONS
- Blood collection was performed for hormone determination from all animals at termination under condition of food deprivation.
- Males: blood samples (approximately 0.8 mL) for hormone determination were collected under isoflurane anaesthesia from the retro-orbital sinus; the order of collection was equalised between groups
- Females: as a part of the necropsy procedure, blood samples (approximately 0.8 mL) for hormone determination were withdrawn from the abdominal vena cava under isoflurane anaesthesia; the order of collection was equalised between groups

BLOOD COLLECTION FOR THYROID HORMONE DETERMINATION (T3, T4 AND TSH)
- Samples were transferred into tubes of appropriate capacity containing no anticoagulant and centrifuged at room temperature
- The serum obtained was divided in two aliquots and stored at -80 °C pending analysis
- Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by a multiplex assay, using Luminex Magpix system and the MILLIPLEX MAP Rat Thyroid Magnetic Bead Panel kit (Merk Millipore, cat. no. RTHYMAG-30K)
- Since no treatment-related effects were seen in the determination performed on Day 14 post partum, samples obtained from females on Day 4 post partum were not analysed and were destroyed

Oestrous cyclicity (parental animals):

- Stock females: oestrous cycle was monitored by vaginal smears in all stock females for 2 weeks before allocation in order to exclude from the study females with irregular cycle
- Females allocated to groups: vaginal smears were taken in the morning starting from Day 1 of dosing up to positive identification of copulation was made.

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 from all females, before despatch to necropsy.

Sperm parameters (parental animals):

- A detailed qualitative examination of the testes was performed in all control and high dose group males killed at term. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.

Litter observations:

STANDARDISATION OF LITTERS
- 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 and 13 post partum.
- Pups killed or dying during the lactation period were weighed before the despatch to necropsy.
- Observation was performed once daily for all litters.
- After culling, all pups were sacrificed with the dams on Day 14 post partum.
- On Day 4 post partum, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment (for example, 5 males and 3 females) was acceptable.
- On Day 4 post partum, at least one culled male and one culled female were selected for hormone determination.
For the following females, the selection was performed on the basis of the pups available:
– Group 1: female no. X1000005 only female pups were selected;
– Group 3: female no. X1000047 only female pups were selected and no. X1000049 only male pups were selected;
– Group 4: female no. X1000065 only female pups were selected; nos. X1000073 and X1000077 only male pups were selected.

PARAMETERS EXAMINED
- 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.
- Body weight of individuals on days 1, 4 and 13 post-partum (pups killed or dying during the lactation period were weighed before the despatch to necropsy)
- Anogenital distance on day 1 post partum (normalised to the cube root of body weight of same day)
- Nipple retention (males) on day 13 post-partum

GROSS EXAMINATION OF DEAD PUPS
- Yes, and some organ weights as detailed below

BLOOD COLLECTION FOR THYROID HORMONE DETERMINATION (T3, T4 AND TSH) on Days 4 and 14 post partum
- On Day 4 post partum, as part of the necropsy procedure, blood samples (approximately 0.2 mL) were taken from 2 pups from each litter (1 male and 1 female, if possible).
- On Day 14 post partum, as part of the necropsy procedure, blood samples (approximately 0.5 mL) were taken from 2 pups from each litter (1 male and 1 female, if possible).
- Blood samples were withdrawn under light ether anaesthesia from the heart (intracardiac puncture). The order of collection was equalised between groups.
- Samples were transferred into tubes of appropriate capacity containing no anticoagulant and centrifuged at room temperature.
- The serum obtained was divided in two aliquots and stored at -80 °C pending analysis.
- Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by a multiplex assay, using Luminex Magpix system and the MILLIPLEX MAP Rat Thyroid Magnetic Bead Panel kit (Merk Millipore, cat. no. RTHYMAG-30K)
- The determination was performed on samples from all pups on Day 14 post partum
- Since no treatment-related effects were seen in the determination performed on Day 14 post partum, samples obtained on Day 4 post partum were not analysed and were destroyed.

Postmortem examinations (parental animals):

EUTHANASIA
- Parental animals that had completed the scheduled test period were killed by exsanguination under isoflurane anaesthesia
- Males were killed after the mating of all females on Day 31 of the study
- Females with live pups were killed on Day 14 post partum; females with total litter loss were killed shortly after the day of the occurrence of total litter loss; females which did not give birth 25 days after positive identification of mating were killed shortly after

NECROPSY
- The clinical history of parental males and females 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 and the required tissue samples preserved in fixative and processed for histopathological examination.
- Females: examined also for the following: external and internal abnormalities; number of visible implantation sites (pregnant animals); number of corpora lutea (pregnant animals). Uteri of females with no visible implantations were immersed in a 10-20 % solution of ammonium sulphide to reveal evidence of implantation

ORGAN WEIGHTS
- From all animals completing the scheduled test period, the following organs were dissected free of fat and weighed and the ratios of organ weight to body weight were calculated for each animal: epididymis (males only); lungs (including mainstem bronchi); ovaries with oviducts (females only); parathyroid glands (weighed and preserved with thyroid gland); prostate gland (dorsal and ventral; males only); seminal vesicles with coagulating glands (males only); spleen; testes (males only); thymus (where present); thyroid; uterus - cervix (females only)

FIXATION PRESERVATION
- Samples of the following 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): abnormalities; adrenal glands; brain (cerebelum, cerebrum, medulla/pons); clitoral gland (females only); epididymis (males only); lungs (including mainstem bronchi); lymph nodes - cervical; lymph nodes - mesenteric; kidneys; liver; mammary gland (both females and males); ovaries with oviducts (females only); seminal vesicles with coagulating glands (males only); testes (males only); thymus (where present); thyroid; uterus - cervix (females only); vagina (females only)

HISTOPATHOLOGICAL EXAMINATION
- The following tissues were required for histopathological examination: abnormalities; adrenal glands; epididymis (males only); lungs (including mainstem bronchi); lymph nodes - cervical; lymph nodes - mesenteric; kidneys; liver; mammary gland (both females and males); ovaries with oviducts (females only); parathyroid glands (weighed and preserved with thyroid gland); pituitary gland; penis (males only); prostate gland (dorsal and ventral; males only); sciatic nerve; seminal vesicles with coagulating glands (males only); spleen; testes (males only); thyroid; uterus - cervix (females only); vagina (females only)
- 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 also performed. The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952 and referred to the comprehensive reviews on the subject: Russell, 1990; Creasy, 1997; Creasy, 2002. The PAS- H stained sections were used to identify the spermatogenic stages. For ease and clarity of reporting, only tissues with findings are reported in the incidence tables.
- The examination was restricted in the first instance as follows: tissues listed from all males and females in the control and high-dose groups killed at term; and all abnormalities in all groups
- On the basis of treatment-related findings detected in the mesenteric lymph nodes and lungs of high dose treated animals of both sexes, the histopathological evaluation of these organs was extended to the low- and mid-dose animals of both sexes.

Postmortem examinations (offspring):

EUTHANASIA
- Pups that had completed the scheduled test period (Day 4 post partum or Day 14 post partum) were euthanised by intraperitoneal injection of Sodium Thiopenthal. Pups selected for blood collection for hormone determination were killed on the day of blood sampling.

NECROPSY
- Pups at Day 4 post partum: all pups found dead in cage were examined for external and internal abnormalities. All culled pups sacrificed on Day 4 post partum were subjected to an external examination. Sex was determined by internal gonads inspection.
- Pups at Day 14 post partum: all live pups sacrificed on Day 14 post partum were examined for external abnormalities and sex confirmation by gonadal inspection. All pups with abnormalities were retained in 10 % neutral buffered formalin.

ORGAN WEIGHTS
- Thyroids were weighed from the one male and one female pups selected for blood collection for hormone determination and preserved in 10 % neutral buffered formalin. The thyroid weights were determined after fixation.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables such as body weight, food consumption, hormone determination and organ weight 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 histopathological findings was 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 was assessed by the nonparametric version of the Williams test. The criterion for statistical significance was p<0.05.

Reproductive indices:

The following reproductive indices were calculated (Developmental and reproductive toxicology: A practical Approach (second edition). Edited by Ronald D. Hood; 2006)
- Males:
Copulation Index (%) = (no.of males with confirmed mating / no.of males cohabitated) × 100
Fertility Index (%) = (no.of males which induced pregnancy / no.of males cohabitated) × 100
- Females:
Copulatory Index (%) = (no.of females with confirmed mating / no.of females cohabitated) × 100
Fertility Index (%) = (no.of pregnant females / no.of females cohabitated) × 100
- Males and females
Pre coital Interval = The number of nights paired prior to the detection of mating

- Females:
Pre-implantation loss was calculated as a percentage from the formula:
((no. of corpora lutea − no.of implantations) / no. of corpora lutea) × 100
Pre-natal loss was calculated as a percentage from the formula:
((no.of visible implantations − live litter size at birth) / no.of visible implantations) × 100
Pup loss at Day 0 post partum was calculated as a percentage from the formula:
((Total litter size − live litter size) / Total litter size) × 100
Post-natal loss at Day 4 post partum (before culling) was calculated as a percentage from the formula:
((Live litter size at birth − live litter size at Day 4, before culling) / Live litter size at birth) × 100
Post-natal loss at Day 13 post partum (after culling) was calculated as a percentage from the formula:
((Live litter size on Day 4(after culling) − live litter size on Day 13) / Live litter size on Day 4 after culling) × 100
Sex ratios were calculated at birth, on Days 4 and 14 post partum and were presented as the percentage of males per litter.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The only clinical sign recorded during the treatment period was black staining on the tail.
This sign was evident in all treated males (slight to marked) and in treated females (slight to moderate) receiving the dose levels of 300 and 1000 mg/kg/day. This discolouration was due to the colour of the test item and its properties.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and body weight gain of treated males were similar to the control animals. Statistically significant decrease in body weight gain was evident in high dose males on Day 15 of treatment. In the following weeks, a trend of recovery was noted in this treated group and at termination body weight gain was comparable to the control group.
Body weight and body weight gain of treated females did not show relevant differences,when compared to the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption was comparable between control and treated groups.

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:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related lesions were noted in mesenteric lymph nodes of high-dose males and females, in the lungs of hig-dose males and in mid- and high-dose females.
At microscopic evaluation, the mesenteric lymph nodes presented minimal presence of macrophages with intracytoplasmic black/yellow/brown granular pigment (pigment laden macrophages) mainly in the superficial cortex and/or medullary cords and/or lymphatic sinuses in all high-dose males and most high-dose females.
Minimal to marked alveolar macrophages with intracytoplasmic black granular pigment, in most occasions associated with chronic inflammatory reactions in the lungs, were observed in most high-dose rats with a predominance among mid- and high-dose females.
The chronic inflammatory reaction was morphologically represented by leukocytes (mainly eosinophils) and macrophages in the perivascular and/or peribronchiolar and/or interstitial compartments of the lung. Minimal intracytoplasmic black granular pigment in the pulmonary alveolar macrophages was only detected in a single male and female (both from the low-dose group). In addition, minimal dark/brown deposits in the epithelium of the mucous membrane of the small intestine tract (ileum) of two high-dose males (gross abnormalities only) was reported.
The remaining sporadic lesions, such as focal necrosis of liver (one high-dose female) or bilateral hypertrophy of adrenals (one high-dose female), were considered to be an expression of spontaneous and/or incidental pathology seen in this species.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

THYROID HORMONES
Statistically significant increases of triiodothyronine and thyroxine were recorded in males of the high-dose group (46 % and 12 %, respectively). Similar increases were also recorded in some animals of the mid- and low-dose groups, with no statistical significance nor clear dose-relation. Since changes were of low severity, with no dose-relation nor concurrent alteration of thyroid stimulating hormone, the above increases were considered to be of no toxicological relevance.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cycle was similar in treated and control groups.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

A detailed qualitative examination of the testes was performed in all control and high dose group males killed at term. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
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 in all control and treated males.

Reproductive performance:

no effects observed

Description (incidence and severity):

One control, one low-dose and one mid-dose dam experienced total litter loss on day 1, day 7 and day 5 post-partum.
One mid-dose and one high-dose dam did not fall pregnant, as determined during the gestation period. Therefore, 9 control dams, 9 low-dose dams, 8 mid-dose dams and 9 high-dose dams had live litters on day 14 post-partum.
Reproductive parameters (pre-coital intervals, copulatory and fertility indices) were similar in treated and control groups.
Gestation periods were similar between treated and control group females: all pregnant females gave birth on Day 22 post coitum (mean value).
The number of corpora lutea, implantation sites, total litter size, pre-implantation loss and pre-natal loss (percentage) was similar in control and treated groups.
All pregnant females gave birth on Day 22 post coitum (mean value). The number of corpora lutea, implantation sites, total litter size, pre-implantation loss and pre-natal loss (percentage) was similar in control and treated groups.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

clinical biochemistry

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive performance

2

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (sperm measures)

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: presence of black pigment laden macrophages in the mesenteric lymph nodes in all high dose males and females, and intracytoplasmic black granular pigment in the alveolar macrophages, associated or not with chronic inflammatory reactions in the lungs

Treatment related:

yes

Dose response relationship:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No signs related to treatment were seen in pups.
Found dead and/or missing pups were recorded in all treated and control groups, particularly evident in females showing a complete loss of their litter (total litter loss). Pups that appeared cold to touch or without food intake (milk) or smaller than others were also noted with similar incidence in all groups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

WHOLE LITTER LOSS
No differences in litter loss were seen between control and treated groups. Two dams (one of the low-dose group and one of the mid-dose group) lost their litters between the day of birth and Days 5 or 7 post partum. In addition, one control female lost its litter on Day 1 post partum.

Body weight and weight changes:

not examined

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

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

No differences in anogenital distance of treated male pups were seen, when compared to the control group.
The mean values of anogenital distance of female pups in order of ascending dose levels were: 1.36, 1.28, 1.36 and 1.25 mm/ 3pg . A slight decrease, significant at statistical analysis, in the mean values was noted in treated females receiving 1000 mg/kg/day, when compared to the control value. However, this change could be considered a positive trend (more “feminisation”), moreover, the value was within the background range.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples were found during the examination of male pups on Day 13 post partum.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Pups thyroid weight on Day 14 post partum: a statistically significant increase in absolute thyroid weight was noted in male pups receiving 100 mg/kg/day. This change was considered of no toxicological significance since it was not dose-related and observed in only one sex.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No findings were noted in culled pups sacrificed on Day 4 post partum and examined externally.
No findings were noted in pups at Day 14 post partum examined externally.
Pup sex determined during the in life phase was confirmed at necropsy by gonads inspection.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

THYROID HORMONES
No changes were observed between the controls and treated pups (day 14 post-partum).

SEX RATIO
Sex ratios (calculated as a percentage of males) at birth and on Days 4 and 14 post partum did not show differences between groups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

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:

viability

sexual maturation

clinical signs

mortality

gross pathology

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Black staining of the tail was the main clinical sign noted during the study in treated animals of both sexes. This sign involved males (all treated groups) more than females (dose level ≥ 300 mg/kg/day).

No differences in body weights, body weight gain or food consumption were recorded in treated animals of both sexes, compared to the control group.

For parental animals, no adverse effects were found on oestrous cycle, copulation and fertility indices, delivery or lactation performance, gestation length, number of corpora lutea or implantation sites. No adverse effects were found in pups such as: anogenital distance, sex ratios, pup weight or pup viability. No treatment-related findings were noted at necropsy.

At macroscopic observations, an increased incidence of dark colour and/or areas of cervical and mesenteric lymph nodes, lungs and intestinal tract (caecum, colon, ileum and rectum) were noted in treated animals of both sexes. An increased incidence of reduced size of thymus was observed in high dose treated females, when compared to the controls.

At microscopic observations, treatment-related lesions were noted in mesenteric lymph nodes of males and females receiving test item at 1000 mg/kg/day, in the lungs of males dosed at 1000 mg/kg/day and in females dosed at ≥300 mg/kg/day.

The findings consisted in the presence of black pigment laden macrophages in the mesenteric lymph nodes in all high dose males and females, and intracytoplasmic black granular pigment in the alveolar macrophages, associated or not with chronic inflammatory reactions in the lungs reported in males dosed at 1000 mg/kg/day and females dosed at ≥300mg/kg/day.

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 300 mg/kg/day for males and 100 mg/kg/day for females.

The NOAEL for reproductive and developmental toxicity was considered to be 1000 mg/kg/day for males and females.

Applicant's summary and conclusion

Conclusions:

The NOAEL for reproductive and developmental toxicity is greater than 1000 mg/kg bw/day.

Executive summary:

The toxicity, as well as, any possible effects of test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of conceptus, parturition and lactation of the offspring, were investigated in an experimental study according to the OECD Guideline 421 (2016). Four groups containing 10 male and 10 female rats each were administered the test item in water softened by reverse osmosis (10 mL/kg of body weight) for 30 days (males) and at least 51 days (females). The test item was given to Sprague Dawley rats by oral administration (gavage) at dosages of 100, 300 and 1000 mg/kg/day. The control group received only the vehicle.

The dosing period for females included (a) a pre-pairing period of two weeks; (b) a pairing phase which lasted between 1 and 4 days; (c) a gestation period of 22 days; and (d) a lactation period of 14 days. Males were dosed for the two-week pre-mating period and pairing phase, then a post-pairing phase; up to a total of 30 days, after which they were sacrificed. All surviving dams with live litters were sacrificed at the end of the study period; females which did not give birth after 25 days of gestation were sacrificed shortly after; dams with total post-natal litter loss were sacrificed shortly after the loss of their litter. At necropsy, all animals were subjected to clinical pathology investigation; gross examination for external/internal abnormalities; number of visible implantation sites (pregnant animals); number of corpora lutea (pregnant animals); thyroid hormone determination (males); terminal body weight; organ weights; histopathological examination; and the copulatory indices and fertility indices were calculated. Litters were adjusted on day 4 post-partum to yield 4 male and 4 female pups per litter; and blood was collected for hormone determination on at least one culled male and one culled female. All surviving pups of the adjusted litters were monitored for sex (at birth), weight (days 1, 4 and 13), anogenital distance (day 1) and nipple retention (day 14) during the post-partum period then were sacrificed on day 14 post-partum. At necropsy, pups found dead were examined for internal/external abnormalities; culled pups were examined externally only; thyroid of one male and one female per dose group was weighed; any abnormalities were preserved.

Mortality and fate of females

No mortality occurred during the study. One female receiving 300 mg/kg/day and one receiving 1000 mg/kg/day were proved not pregnant at necropsy. A total of 3 females, one control, one receiving 100 and one receiving 300 mg/kg/day had total litter loss on Days 1, 7 and 5 post partum, respectively. The control females with total litter loss showed also unilateral implantation at necropsy. The number of females with live pups on Day 14 post partum was: 9 each in the control, low dose (100 mg/kg/day) and high dose (1000 mg/kg/day) group and 8 in the mid-dose dose group (300 mg/kg/day).

Clinical signs

The only clinical sign recorded during the treatment period was black staining on the tail (slight to marked) in all treated males and in females (slight to moderate) receiving the dose level ≥300 mg/kg/day.

Body weight and body weight gain

No adverse effects were observed in body weight or body weight gain of treated animals compared to control group.

Food consumption

Food consumption of parental animals of both sexes was unaffected by treatment.

Thyroid hormones

Parental males

Statistically significant increases of triiodothyronine and thyroxine were recorded in males dosed at 1000 mg/kg/day (46 % and 12 %, respectively). Similar increases were also recorded in some animals receiving 100 and 300 mg/kg/day, with no statistical significance nor clear dose-relation. Since changes were of low severity, with no dose-relation nor concurrent alteration of thyroid stimulating hormone, the above increases were considered to be of no toxicological relevance.

Pups - Day 14 post partum

No changes were observed between the control and treated pups

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 sites, pre-implantation loss data, pre-natal loss dataandgestation length of females

No significant differences were observed for these parameters between the treated groups and controls. All pregnant dams gave birth on Day 22 post coitum (mean value).

Litter data at birth, on Days 1 and 4 post partum (before culling) and on Day 13 post partum (after culling) and sex ratio of pups

Litter data and sex ratio were unaffected by treatment, at birth, on Days 1, 4 and 14 postpartum.

Clinical signs of pups

No compound-related effects were observed. Pre-weaning clinical signs were comparable between treated and control groups or considered incidental.

Anogenital distance and nipple count

No treatment-related effects were seen both in males and in female pups compared to controls.

No nipples were present in males on Day 13 post partum.

Necropsy findings in decedent pups, in pups sacrificed on Days 4 and 14 post partum

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 weight on Day 14 post partum

No treatment-related changes were seen between control and treated groups.

Terminal body weight and organ weights

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

Macroscopic observations

An increased incidence of dark colour and/or areas of cervical and mesenteric lymph nodes, lungs and intestinal tract (caecum, colon, ileum and rectum) were noted in treated animals of both sexes. An increased incidence of reduced size of thymus was observed in high dose treated females, when compared to the controls.

Microscopic observations and spermatogenic cycle

Treatment-related lesions were noted in mesenteric lymph nodes of males and females receiving test item at 1000 mg/kg/day, in the lungs of males dosed at 1000 mg/kg/day and in females dosed at ≥300 mg/kg/day.

The findings consisted in the presence of black pigment laden macrophages in the mesenteric lymph nodes in all high dose males and females, and intracytoplasmic black granular pigment in the alveolar macrophages, associated or not with chronic inflammatory reactions in the lungs reported in males dosed at 1000 mg/kg/day and females dosed at ≥300 mg/kg/day.

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 in all control and treated males.

Conclusion

The NOAEL for reproductive and developmental toxicity was considered to be 1000 mg/kg/day for males and females.