Cuprate(6-), [2-[[[[3-[[4-chloro-6-[[4-[[4-chloro-6-[(3-sulfophenyl)amino]-1,3,5-triazin-2-yl]amino]-2,5-disulfophenyl]amino]-1,3,5-triazin-2-yl]amino]-2-hydroxy-5-sulfophenyl]azo]phenylmethyl]azo]-5-sulfobenzoato(8-)]-, pentasodium hydrogen, (SP-4-3)-

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

The NOAEL (No Observed Adverse Effect Level) for general toxicity and reproductive and developmental toxicity was considered to be 1000mg/kg body weight/day for both males and females.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of experimental phase (Allocation of animals): 16 July 2019 End of experimental phase (Last day of necropsy): 15 October 2019 Study completion: Date of Study Director’s signature on the report

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted on 29 July 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat was 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:

- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10-11 weeks main groups/ 13-14 weeks recovery groups
- Weight at study initiation: At start of dosing, approximately 410 g for males and 270 g for females
- Fasting period before study: no
- Housing: From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulfone
solid bottomed cages measuring 59.5×38×20 cm. During mating, animals were housed 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. After mating, the males were re-caged as they were before mating. The females were tra
nsferred to individual solid bottomed cages measuring 42.5×26.6×18.5 cm for the gestation period,
birth and lactation. Nesting material was provided inside suitable bedding bags. In addition, suitable
nesting material (Scobis 0Mucedola) was provided as necessary and changed at least 2 times a wee
k.
- Diet (e.g. ad libitum): 4 RF 21, Mucedola S.r.l.; ad libitum except when bleeding was performed
- Water (e.g. ad libitum): Drinking water was supplied ad libitum to each cage via water bottles
- Acclimation period (From arrival): An acclimatisation period of 26 days (main groups) and 40 days
(recovery groups) was allowed before the start of treatment
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): the rooms were lit by artificial light for 12 hours each day
IN-LIFE DATES: From: 16 Luglio 2019 (allocation to the study) To: 15 Ottobre 2019 (Last day
of necropsy)

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

softened water (by reverse osmosis)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was dissolved in the vehicle. The preparations were
made weekly at concentrations of 10, 30 and 100 mg/mL. Concentrations were calculated
and expressed in terms of test item as supplied.


VEHICLE: softened water (by reverse osmosis)
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw

Details on mating procedure:

Mating was monogamous (one male to one female). Vaginal smears were 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 of copulation occurred.

Vaginal smears were taken in the morning from Day 1 of treatment, up to positive identification
of mating including not less than 2 weeks before the pairing.
The vaginal smear data were examined to determine the following:
– anomalies of the oestrous cycle;
– 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the preparations made on two occasions during the study (Week 1 and again towards the
end of the study) were analysed to check the concentration. Results of the analyses were within the
acceptability limits stated in ERBC SOPs for concentration of solutions (90-110%).

Duration of treatment / exposure:

Main groups (Groups 1 to 4):
Males
Animals were dosed for 2 consecutive weeks prior to pairing, through the mating period and thereafter
for a total of 34/35 days.
Females
Animals were dosed for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum
and post partum periods until Day 13 post partum (for at least 51 days of total treatment period). The
non pregnant females were dosed up to the day before necropsy.
Recovery groups (Groups 5 and 6)
Males were dosed for 28 consecutive days. Females were dosed for up to 8 consecutive weeks (total
of 56 days).

Frequency of treatment:

Animals were dosed once a day, 7 days a week

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

low dose - main group

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

mid-dose - main group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

high dose - main and recopvery

No. of animals per sex per dose:

Main groups (Groups from 1 to 4): 10 male and 10 female rats
Recovery groups (Groups 5 and 6): 5 male and 5 female rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels of 100, 300 and 1000 mg/kg body weight/day were selected
based on information from a previous study. In an acute oral toxicity study in rats, at dose levels of 2000 and 4000 mg/kg body weight (test item purity 71.2%), no deaths occurred. No signs of toxicity were observed at 2000 mg/kg body weight. Slight piloerection and hunched posture were observed within the first 24 hours after administration at dose level of 4000 mg/kg body weight.

- Rationale for animal assignment (if not random): computerised stratified randomisation to give approximately equal initial group mean body weights
- Fasting period before blood sampling for clinical biochemistry: yes
- Rationale for selecting satellite groups: Usually with dyes, reversible staining of organs and tissues is noted, as well as re-absorption of excreted dye-material in the kidney tubules with storage of dye material in lysosomes. Due to the high amount of salt, local irritation in the stomach mucosa is often noted as well. Therefore, a recovery group in the high-dose and control group is included to check reversibility of apparent effects.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): random

Parental animals: Observations and examinations:

Mortality (All groups)
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 groups)
Before commencement of treatment and at least once daily during the study, each animal was obs
erved and any clinical signs were recorded. Observations were performed at the same time interval e
ach day, the interval was selected taking into consideration the presence of post-dose reactions (2-2.5
hour after treatment).

Observations of the cage tray
Observations of the cage tray during the pre-mating, after mating (for males) and post partum periods
were performed for all groups and were recorded three times weekly (main groups).
During mating (males and females) and gestation periods (females), these observations were
performed for all main groups and were recorded daily.
Observations of the cage tray for recovery groups were performed, during both treatment and recov
ery periods, three times weekly.

Clinical Observations (Functional Observation Battery Tests) (All groups)
Once before commencement of treatment and at least once a week from the start of treatment until te
rmination, each animal was given a detailed clinical examination. Each animal was removed from the
home cage and observed in an open arena. The tests included observation of changes in gait and
posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behavio
ur and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusualrespiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and
excretions were also recorded. All observations were recorded for individual animals.
For the main groups, data are reported untilWeek 5 of study (males) and untilWeek 6 (when all
females were present). For the recovery groups, data are reported during treatment until Week 4 for
males and 8 for females andWeek 2 of recovery.

Grip strength and sensory reactivity to stimuli (All groups)
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected
from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, vi
sual and proprioceptive stimuli) and for assessment of grip strength. Measurements were performed u
sing a computer generated random order (for the main groups only).
In the main groups, the tests were performed on Day 30 of the study for males and on Day 12 post
partum for females.
In the recovery groups, the tests were performed during treatment period on Day 22 for males and 51
for females and again duringWeek 2 of recovery in all animals.

Motor activity assessment (MA) (All groups)
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected
from each group and the motor activity was measured (for approximately 5 minutes) by an a
utomated activity recording device. Measurements were performed using a computer generated
random order (for the main groups only).
In the main groups, the tests were performed on Day 29 of the study for males and on Day 12 post
partum for females.
In the recovery groups, the tests were performed during treatment period on Day 22 for males and 51
for females and again duringWeek 2 of recovery in all animals.

Body weight (All groups)
Main groups
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.
Recovery groups
Each animal was weighed on the day of allocation to treatment groups, on the day that treatment
commenced, weekly thereafter and just prior to necropsy. Body weights of recovery animals on the
day of allocation are not tabulated in this report, but will be archived with all raw data.

Food consumption (All groups)
Main groups
The weight of food consumed by each cage of males and females was recorded weekly during the
pre-mating period starting from Day 1 of dosing. 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.
Recovery groups
The weight of food consumed by each cage of rats was recorded at weekly intervals starting from Day
1 of dosing.

Clinical pathology investigations
Blood samples for haematology, clinical chemistry and coagulation were collected, at the end of trea
tment period, by random selection from 5 males and 5 females (females with viable litters) of each
main group. Further blood samples for haematology, clinical chemistry and coagulation were taken un
der identical conditions at the end of the recovery period.
Males
Blood samples for haematological investigations, biochemical tests and hormone determination wer
e collected in condition of food deprivation under isoflurane anaesthesia from the retro-orbital sinus.
Blood samples for coagulation test (food available) were collected at necropsy from the vena cava
under isoflurane anaesthesia. The order of collection was equalised between groups.
Females
As a part of the sacrificial procedure, blood samples for all determinations were withdrawn from the
abdominal vena cava in condition of food deprivation under isoflurane anaesthesia. The order of coll
ection was equalised between groups.
The blood samples were divided into tubes as follows:
– EDTA anticoagulant for haematological investigations
– Heparin anticoagulant for biochemical tests
– Citrate anticoagulant for coagulation tests
– No anticoagulant for hormone determination

Blood collection and thyroid hormone determination (T3, T4 and TSH) (Main groups)
Blood collection from parental animals
Blood collection was performed for hormone determination (0.8 mL) from all parental animals at term
ination under condition of food deprivation.
Blood collection from pups 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 (1 male and 1 female, where possible).
On Day 14 post partum as part of the necropsy procedure, blood samples (approximately 0.5 mL) w
ere taken from 2 pups (1 male and 1 female).
Blood samples were withdrawn under light ether anaesthesia from the heart (intracardiac puncture).
The order of collection was equalised between groups.

Parental animals and pups
Samples were transferred into tubes containing no anticoagulant and centrifuged at roomtemp
erature. The serum obtained was divided in two aliquots and stored at -80°C pending analysis (secti
on 4.4.4).

Bioanalysis - Thyroid hormone determination (T3, T4 and TSH) (Main groups)
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
LuminexMagpix system and the MILLIPLEX MAP Rat Thyroid HormoneMagnetic Bead Panel kit
(MerkMillipore, cat. no. RTHYMAG-30K).
The determination was restricted as detailed below:
– Samples from all parental males from all groups
– Samples from pups on Day 14 post partum
Since no treatment-related effects were seen in the determination performed in parental males and in
pups on Day 14 post partum, samples obtained from females and from pups on Day 4 post partum
were not analysed and will be returned to the Sponsor.
When the serum levels for Total triiodothyronine (total T3) were BLOQ (below the limit of
quantification - 0.625 ng/mL), the values were not included in the mean calculation.

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. These data are
not tabulated in this report, but will be archived with all raw data.

Females allocated to groups
Vaginal smears were taken in the morning from Day 1 of treatment, up to positive identification
of mating including not less than 2 weeks before the pairing.
The vaginal smear data were examined to determine the following:
– anomalies of the oestrous cycle;
– 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 five randomly selected 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 treatmentrelated 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.

Litter observations:

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 dying during the lactation period were weighed before the despatch to necropsy.
Observations were performed once daily for all litters.
After culling, all pups were sacrificed with the dams on Day 14 post partum.

Anogenital distance (AGD) (Main groups)
The anogenital distance (AGD) of each pup was measured on Day 1 post partum. The measure of AGD was normalized to the cube root of the pup’s body weight measured on Day 1 post partum.

Nipple count (Main groups)
The presence of nipples/areolae in male pups was checked on Day 13 post partum (a day before despatch to necropsy).

Postmortem examinations (parental animals):

Euthanasia (All groups)
Parental or recovery animals that had completed the scheduled test period, were killed by exsanguination under isoflurane anaesthesia.

Parental males (Main groups)
The males were killed after the mating of all females on Days 35 and 36 of the study.

Parental females (Main groups)
The females with live pups were killed on Day 14 post partum. The females which did not give birth 25 days after positive identification of mating or with total litter loss were killed shortly after.

Males and females (Recovery groups)
Animals were killed at the end of the 2-week recovery period.

Necropsy (All groups)
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 and the required tissue samples preserved in fixative and processed for histopathological examination.
Females
Parental females were examined also for the following:
– number of visible implantation sites (pregnant animals);
– number of corpora lutea (pregnant animals).
Uteri of apparently non-pregnant females were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

Organ weights (All groups)
Parental animals
From all animals completing the scheduled test period, the organs indicated in section 4.5.7 of the report were dissected free of fat and weighed.
The ratios of organ weight to body weight were calculated for each animal.

Tissues fixed and preserved (All groups)
Samples of all the tissues listed in section 4.5.7 of the report 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
The tissues required for histopathological examination are listed in section 4.5.7 of the report. 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.

The histopathological examination was restricted as detailed below:
i Tissues specified in section 4.5.7 of the report from 5 males and 5 females randomly selected (animals evaluated for clinical pathology with the exception of two females of the control group) in the control and high dose main group killed at term.
ii All abnormalities in main groups.

Postmortem examinations (offspring):

Euthanasia (All groups)
Pups
Pups that had completed the scheduled test period (Day 4 or Day 14 post partum) were euthanised by intraperitoneal injection of Sodium Thiopenthal.

Necropsy (All groups)
Pups
All pups found dead in the 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.
All live pups sacrificed on Day 14 post partum were killed and examined for external abnormalities and sex confirmation by gonadal inspection.
Pups with abnormalities were retained in 10% neutral buffered formalin.

Nipples retention at Day 14 post partum
The nipples/areolae in male pups, if recorded on Day 13 post partum were retained (mammary area) in a 10% neutral buffered formalin.

Organ weights (All groups)
Pups at Day 14 post partum
Thyroids were weighed from one male and one female pup selected for blood collection for hormones determination and preserved in 10% neutral buffered formalin. The thyroid weights were determined after fixation.

Statistics:

Standard deviations were calculated as appropriate. For variables, e.g. body weight, food
consumption, clinical pathology parameters and organ weights, 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 was used for the other parameters.
Intergroup differences between the control and treated groups were assessed by the nonparametric
version of theWilliams test. The criterion for statistical significance was p<0.05.
The mean values, standard deviations and statistical analysis were calculated from actual
values in the computer without rounding off.

Reproductive indices:

Group mean values were calculated for all parameters. Data from non-pregnant female were
excluded from group mean calculations as considered appropriate by the Study Director.
Males
Copulatory 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 =Number of nights paired prior to the detection of mating

Offspring viability indices:

Pre-implantation loss was calculated as a percentage from the formula:
no. of corpora lutea−no. of implantation/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

Post natal 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 fromthe 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 fromthe formula:
live litter size onDay 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 Day 4 and Day 14 post partum and were presented asthe percentage of males per litter.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Main and recovery groups
Blue staining in different regions of the body surface (related to the nature and intended use of the
test item) was observed during the study, in the high dose group only. Other clinical signs observed,
such as scabs, hairloss and damaged eye, were generally observed in a limited number of animals
and were not considered related to the treatment.
Observations of the cage tray
– Blue staining on the cage tray and blue faeces were observed during the pre-mating and mating per
iods in animals of mid- and high dose of main groups of both sexes and after mating in high dose ma
les up to the end of treatment.
– During the gestation period, blue faeces were observed only in the first days of the gestation period
in the mid-dose females while blue staining and blue faeces on the cage tray were noted in the high
dose females during the entire gestation period.
– Blue faeces on the cage tray was observed in females of the high dose group during the post
partum period.
– During the dosing phase, recovery group (males and females) showed blue staining and blue faeces
on the cage tray. These signs disappeared immediately after cessation of treatment in females and
from Day 2/3 of the recovery phase in males.
The above mentioned findings were considered related to the colour of the test item which was
eliminated by the animals.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred throughout the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Main groups
No relevant differences in body weight and body weight gain were noted between control and treated
males and females throughout the study.
Recovery groups
Means of body weight and body weight gain were comparable between controls and the treated grou
ps both in males and females throughout the treatment and recovery phase.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Main and recovery groups
Food consumption was unaffected by treatment in both sexes during the study.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Dosing phase -Main groups
No relevant changes attributable to the treatment were noted.
The statistically significant differences of eosinophils recorded between control and females dosed at
300mg/kg body weight/day (-60%) was not dose-related, therefore it was considered to be unrelated
to treatment.
Recovery phase - Recovery groups
The statistically significant decreases of mean corpuscular haemoglobin and mean corpuscular hae
moglobin concentration observed in treated males at the end of the recovery phase were not seen at
the end of treatment of main group animals, therefore they were considered to be incidental.
Coagulation
Dosing phase -Main group
No treatment-related effects were recorded in prothrombin time measured at the end of the study.
Recovery phase - Recovery groups
Prothrombin time was statistically significantly higher than controls in treated females (4%). Since
this finding was not recorded at the end of the dosing period in main group females, this change was
considered to be unrelated to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Dosing phase -Main groups
Alanine aminotransferase was decreased in males dosed at 1000mg/kg body weight/day (34% belo
w controls). The decrease of such parameter usually has no pathological significance, therefore this
finding was considered to be not adverse.
The other statistically significant differences between control and treated animals (chloride in males,
aspartate aminotransferase and urea in females) were not dose-related, therefore they were cons
idered to be unrelated to treatment.
Recovery phase - Recovery groups
Glucose was statistically significantly higher than controls in treated males (18%). The same effect
was also noted at the end of dosing in main group animals, even though the differences to control
males were not statistically significant. In both cases, changes were of minimal severity and withou
t dose-relationship, therefore they were considered to be not adverse. The statistically significant
increases of gamma-glutamyl transferase and globulin observed in treated femaleswere not recorded
at the end of treatment, therefore theywere considered to be incidental.

Endocrine findings:

no effects observed

Description (incidence and severity):

Parental males
Thyroid stimulating hormone and triiodothyronine were increased in one male dosed at 1000mg/kg
body weight/day (no. 70). Compared with mean control data, the increases were 5.0 and 2.8 fold,
respectively. The simultaneous increase of these two hormones usually has no pathological signifi
cance, therefore this change was considered to be not adverse.
Pups on Day 14 post partum
No changes were seen in thyroid hormones of pups sacrificed on Day 14 post partum.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Clinical observations (Functional Observation Battery Tests)
Main and recovery groups
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did
not reveal changes attributable to the test item.
Motor activity and sensory reaction to stimuli
Main and recovery groups
No significant alterations in motor activity, grip strength or sensory reaction to stimuli were observed
in any treatment group at the examinations performed during the study. The statistically significan
t increase in grip strength (first, second trial and mean) noted at the end of treatment in high dose
males of the recovery group was not attributed to the test item but considered due to the lower body
weight at the time of test when compared with control.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Final sacrifice
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: oestrous cycle:

no effects observed

Description (incidence and severity):

The total number of oestrous cycles observed in all females before pairing (number of non
sequential days in which the females were in oestrous) were similar between control and
treated groups and was of 3-4 times.
Vaginal smears examined on the day of necropsy to determine the stage of the oestrous
cycle showed the phase of diestrous for the not pregnant control females (nos. 3, 17 and 19)
and that of diestrous for the majority of females sacrificed on Day 14 post partum.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

All females mated within 1-4 days of cohabitation.
The number of copulatory plugs found in the cage were 4 (mean value) for all groups.
Copulatory index was 100% for both males and females of all treated groups. Fertility
index both for males and females was 100% each for all treated groups and 70% for control
group. Three control males (nos. 4, 18 and 20) failed to induce pregnancy. These cases were
considered incidental.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No effects related to treatment were seen.
Apparently no food intake (milk), small appearance, pallor and cold to touch were the main
clinical signs noted in control and treated pups.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Found dead and/or missing pups were observed both in control and treated groups, with
similar incidence.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Litter data of treated females (e.g. total litter size, live litter size, pup loss, litter weight and
mean pup weight) did not show dose-related or treatment-related differences.

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No relevant differences were noted in thyroid weight of pups between control and treated
groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Necropsy findings in pups

Deceased pups
At macroscopic observations, autolysed abdominal organs were observed in the majority of deceased pups, both in control and treated groups.

Pups sacrificed on Days 4 and 14 post partum
No findings were recorded in pups sacrificed on Day 4 post partum (culled pups).
No toxicologically relevant or treatment related findings were recorded in pups sacrificed on Day 14 post partum. For one dam only (no. 45 - Group 3), hairloss on the dorsum was seen in all pups.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level)
for general toxicity and reproductive and developmental toxicity was considered to be
1000mg/kg body weight/day for both males and females.

Executive summary:

The toxic effects on Sprague Dawley rats after repeated oral dosing with Reactive Blue 160, were investigated in combined repeat-dose toxicity study with reproduction/developmental toxicity screening. A 2-week treatment-free period was included in order to assess any delayed toxicity or recovery from any adverse effects observed during the dosing phase. The dose levels used during the study were: 0, 100, 300 and 1000 mg/kg body weight/day. Recovery groups received 0 and 1000 mg/kg body weight/day.

There was no mortality during the study. During the in vivo phase of the study, no treatment-related signs of toxicity were seen in clinical signs, neurotoxicity assessment, body weight and food consumption. Blue staining in different regions of the body surface and blue staining and blue faeces in litter tray were observed during the study, this abnormal colouration was due to the nature and intended use of the test item (dye).

Changes observed in some haematological and coagulation parameters (eosinophils, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration, prothrombin time) were considered incidental or unrelated to the treatment. Fluctuations in alanine aminotransferase and glucose in males treated at 1000 mg/kg body weight/day were considered to be not adverse. Increase in thyroid stimulating hormone and triiodothyronine in a single male dosed at 1000 mg/kg body weight/day was considered to be an incidental effect and not adverse.

The total number of oestrous cycles observed in all females before pairing and pre-coital intervals, copulatory and fertility indices did not show any differences that could be related to treatment. No treatment-related differences were noted in implantation sites, pre-natal loss data and gestation length of females between treated and control groups, as well as in litter data and sex ratios.

Pre-weaning clinical signs in pups were unaffected by the treatment. No nipples were observed in male pups on Day 13 post partum. Thyroid weight recorded in treated pups was comparable to the control mean value. No changes were observed in thyroid hormones measured in pups on Day 14 post partum. No treatment-related effects were observed in anogenital distance measured in male and female pups of the treated groups.

No toxicologically relevant changes were observed in terminal body weight and in organ weights of treated animals that completed the treatment or recovery periods. Necropsy findings in deceased pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect. The most relevant changes observed at necropsy performed at the end of the treatment periods in parental animals were blue colour of gastrointestinal tract, urogenital tract or staining of skin, were due to the colour and nature of the test item. No remarkable differences were noted at post mortem examinations at the end of the recovery period.

At the histopathological examination (5/animals/sex of control and high-dose main groups), no treatment-related changes were noted. In addition, regular layering in the germinal epithelium was noted in 5 males of control and high-dose main groups.

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity and reproductive and developmental toxicity was considered to be 1000 mg/kg body weight/day for both males and females.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The toxic effects on Sprague Dawley rats after repeated oral dosing with Reactive Blue 160, were investigated in combined repeat-dose toxicity study with reproduction/developmental toxicity screening. A 2-week treatment-free period was included in order to assess any delayed toxicity or recovery from any adverse effects observed during the dosing phase. The dose levels used during the study were: 0, 100, 300 and 1000 mg/kg body weight/day. Recovery groups received 0 and 1000 mg/kg body weight/day.

There was no mortality during the study. During the in vivo phase of the study, no treatment-related signs of toxicity were seen in clinical signs, neurotoxicity assessment, body weight and food consumption. Blue staining in different regions of the body surface and blue staining and blue faeces in litter tray were observed during the study, this abnormal colouration was due to the nature and intended use of the test item (dye).

Changes observed in some haematological and coagulation parameters (eosinophils, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration, prothrombin time) were considered incidental or unrelated to the treatment. Fluctuations in alanine aminotransferase and glucose in males treated at 1000 mg/kg body weight/day were considered to be not adverse. Increase in thyroid stimulating hormone and triiodothyronine in a single male dosed at 1000 mg/kg body weight/day was considered to be an incidental effect and not adverse.

The total number of oestrous cycles observed in all females before pairing and pre-coital intervals, copulatory and fertility indices did not show any differences that could be related to treatment. No treatment-related differences were noted in implantation sites, pre-natal loss data and gestation length of females between treated and control groups, as well as in litter data and sex ratios.

Pre-weaning clinical signs in pups were unaffected by the treatment. No nipples were observed in male pups on Day 13 post partum. Thyroid weight recorded in treated pups was comparable to the control mean value. No changes were observed in thyroid hormones measured in pups on Day 14 post partum. No treatment-related effects were observed in anogenital distance measured in male and female pups of the treated groups.

No toxicologically relevant changes were observed in terminal body weight and in organ weights of treated animals that completed the treatment or recovery periods. Necropsy findings in deceased pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect. The most relevant changes observed at necropsy performed at the end of the treatment periods in parental animals were blue colour of gastrointestinal tract, urogenital tract or staining of skin, were due to the colour and nature of the test item. No remarkable differences were noted at post mortem examinations at the end of the recovery period.

At the histopathological examination (5/animals/sex of control and high-dose main groups), no treatment-related changes were noted. In addition, regular layering in the germinal epithelium was noted in 5 males of control and high-dose main groups.

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity and reproductive and developmental toxicity was considered to be 1000 mg/kg body weight/day for both males and females.