Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 03 Oct to 09 Dec 2014
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)
Deviations:
yes
Remarks:
one genetox endpoint was included
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy
- Age at study initiation: 7 to 8 weeks (day of allocation)
- Weight at study initiation: (P) Males: 251 g; Females: 213 g
- Fasting period before study: no
- Housing: 5 animals/sex/cage
- Diet (e.g. ad libitum): ad libitum except for clinical pathology
- Water (e.g. ad libitum): ad libitum except for clinical pathology
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 2°C
- Humidity (%): 55%+/-15%
- Air changes (per hr): approximately 15 to 20 air change per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: from 03 October (day of allocation) to 09 December 2014 (last day of necropsy)
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: up to 7 days interval


VEHICLE
- Justification for use and choice of vehicle (if other than water): n/a
- Concentration in vehicle: 6.25, 25, 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): n/a
- Purity: n/a
Details on mating procedure:
- M/F ratio per cage: one male to one male
- Length of cohabitation: up to 14 days ( 2 sessions of mating)
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): single
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable. Samples of the formulations prepared during the study, on the first and last week of treatment (when all females were present) were also analysed to check the concentration. In addition, in the present study a 8 day stability at +4°C was verified at 6.25 mg/mL. The overall results of the analyses were within the limits of acceptance stated in RTC SOPs for concentration (90-110%).

Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. A0001) by a spectrophotometer analysis. The software used for this activity was Skanlt® version 2.4.2.55 (Thermo Scientific).
Duration of treatment / exposure:
Males
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, through the mating period and thereafter
until the day before necropsy for a total of 48 days.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and
post partum periods until at least up to, and including, Day 3 post partum or the day before sacrifice. Dose volumes were adjusted once per week for
each animal according to the last recorded body weight. One female, no. 33 (Group 2), was erroneously dosed on Day 4 post partum.
During the gestation period, dose volumes were calculated according to individual body weights on Days 0, 7, 14 and 20 post coitum and on Day 1
post partum. Thereafter individual dose volumes remained constant.

Recovery groups
Animals were dosed once a day, 7 days a week, for a minimum of 4 consecutive weeks. No treatment was given during the recovery period.
Frequency of treatment:
Once a day
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Main groups
3 groups of 10 males and 10 females each dosed at 62.5, 250 and 1000 mg/kg/day bw. One control group (group 1) of 10 males and 10 females
received the vehicle alone.

Recovery groups
1 group of 5 males and 5 females dosed at 1000 mg/kg/day bw. One control group (group 5) of 5 males and 5 females received the vehicle alone.
Control animals:
yes, concurrent vehicle
Details on study design:
The Sprague Dawley SD rat was the species and strain of choice because it is accepted by many regulatory authorities for reproductive toxicology studies and there is ample experience and background data on this species and strain. The test item was administered orally by gavage to parental animals at 10 mL/kg body weight. Dose levels of 62.5, 250 and 1000 mg/kg/day were selected by the Sponsor based on information from previous studies. The oral route was selected as it is a possible route of exposure of the test item in man.
Animals were randomly assigned to groups weighted by body weights to assure a similar mean body weight in each group.
Parental animals: Observations and examinations:
MORTALITY
Throughout the study, all animals were checked 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, each animal of the main and recovery groups, was observed and
any clinical signs were recorded.
Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose
reactions.
Once before commencement of treatment and at least once daily during the study each animal of the positive control group was observed and any
clinical signs were recorded. These data are not not presented in this report but retained and archived as study raw data.

CLINICAL OBSERVATIONS (FUNCTIONAL OBSERVATION BATTERY TESTS)
Once before commencement of treatment and at least once per week from the start of treatment, each animal was given a detailed clinical examination.
Each animal was observed in an open arena. The test included observation of changes in gait and posture, reactivity to handling, presence of clonic
or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual
respiratory pattern).
Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded.
All observations were recorded for individual animals.

MOTOR ACTIVITY
Once 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 automated activity recording device. Measurements were performed using a computer generated random order (for the main groups). For males the tests were performed 7 days before necropsy and for females on Day 3 post partum. Once during Week 4 of recovery, the
motor activity was also performed for all animals.

GRIP STRENGTH AND SENSORY REACTIVITY TO STIMULI
Once towards the end of treatment, 5 males and 5 females were selected from each group for evaluation of sensory reactivity to stimuli of different
modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. Measurements were performed using a computer
generated random order (for the main groups). For males the tests were performed 6 days before necropsy and for females on Day 3 post partum.
Once during Week 4 of recovery, these evaluations were also performed in all animals.

BODY WEIGHT
Main groups
Males were weighed weekly from allocation to termination. Body weights measured at term (days 34 and 35) are not presented in this report, but
retained and archived as study raw data. Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0,
7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum.
For females, body weights measured weekly during the mating phase are not presented in this report but retained and archived as study raw data.

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.


FOOD CONSUMPTION
Main groups
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 gestation Days 7, 14 and 20 starting from Day 0 post coitum and
on Day 4 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 following allocation.

BLOOD CLOTTING TIME
During the last week of treatment, a measure of blood clotting time was performed once on the same 5 males and 5 females randomly selected from each group as specified in Clinical-pathology section (only main groups).

CLINICAL PATHOLOGY
As a part of the sacrificial procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males
and 5 females (females with viable litters) randomly selected from each group, under condition of food deprivation. At the end of
Week 4 of the recovery period, blood samples were also taken from all surviving animals under identical conditions in order to re-evaluate
haematology parameters, gamma-glutamyltransferase and bilirubin which showed treatment-related changes at measurements performed
during the treatment period.

The blood samples collected were divided into tubes as follows:
EDTA anticoagulant fro haematological investigations
Heparin anticoagulant for biochemical tests
citrate anticoagulant for coagulation tests

The measurments performed on blood samples are listed below:
Haematocrit
Haemoglobin
Red blood cell count
Reticulocyte count
Mean red blood cell volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
White blood cell count
Differential leucocyte count
– Neutrophils
– Lymphocites
– Eosinophils
– Basophils
– Monocytes
– Large unstained cells

Platelets

Coagulation
- Prothrombin time
- Activated partial thromboplastin time
- Blood clotting time (in vivo test)

Clinical chemistry
Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Gamma-glutamyltransferase
Urea
Creatinine
Glucose
Triglycerides
Bile acids
Inorganic phosphorus
Total bilirubin
Total cholesterol
Total protein
Albumin
Globulin
A/G Ratio
Sodium
Potassium
Calcium
Chloride

Urinalysis (only males) (main groups)
At the same time interval of the clinical pathology investigations, individual overnight urine samples were also collected from the same animals
under the same conditions. Before starting urine collection, water bottles were removed from each cage and each animal received approximately
10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis.
The measurements performed on the urine samples are listed below:
Appearance
Volume
Specific gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Blood

The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for:

Epithelial cells
Leucocytes
Erythrocytes
Crystals
Spermatozoa and precursors
Other abnormal components
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning starting two weeks before pairing throughout the mating period, until a positive identification of copulation was made.
The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight, prostate weight, seminal vesicles weight, morphological evaluation of the seminiferous epithelium (staging of the spermatogenic cycle)
Litter observations:
As soon as possible, after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups
were identified. Live pups were individually weighed on Days 1 and 4 post partum. Pups killed or dying during the lactation period were weighed
before the despatch to necropsy. Observations were performed once daily for all litters.
Postmortem examinations (parental animals):
Parental animals that had completed the scheduled test period and selected for blood collection, were killed by exsanguination under isofluorane anaesthesia. Animals that had completed the scheduled test period and not selected for blood collection, were killed by carbon dioxide asphyxiation.

Pups were euthanised by intraperitoneal injection of Thiopenthal.

Parental males (Main groups)
The males were killed after the mating of all females. The total treatment period was up to 48 days.

Parental females (Main groups)
The females with live pups were killed on Day 4 post partum. The females which did not give birth 25 days after positive identification of mating
were killed shortly after.

Males and females (Recovery groups)
Animals were killed after 4 weeks of recovery.

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 (Main groups)
All females were examined also for the following:
1. number of visible implantation sites (pregnant animals);
2. number of corpora lutea (pregnant animals).
Uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

Organ weights – Main and recovery groups
From all animals completing the scheduled test periods, the organs were dissected free of fat and weighed.
The ratios of organ weight to body weight were calculated for each animal.

Tissues fixed and preserved – Main and recovery groups
Samples of all the tissues listed 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 – Main and recovery groups
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 examination was restricted as detailed below:

1. Tissues from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term.
2. All abnormalities in all main groups.
Postmortem examinations (offspring):
All pups found dead in the cage and one pup sacrificed for humane reasons were examined for external and internal abnormalities.
The sex of pup sacrificed for humane reasons was defined at necropsy. All live pups sacrificed at termination were killed and examined for external abnormalities and sex confirmation by gonadal inspection. No pups with abnormalities were retained.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means were assessed
by Dunnett’s test or a modified t test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance was used for the
other parameters. The criterion for statistical significance was p < 0.05.
Reproductive indices:
Males
Copulatory Index (%)=no. of animals mated/no. of animals x100
Fertility Index (%)=no. of males which induced pregnancy/no. of males paired x100

Females
Copulatory Index (%)=no. of animals mated/no. of animals x100
Fertility Index (%)=no. of prenant females/no. of females paired x100

Males and Females
Pre-coital Interval=Mean number of days between pairing and mating
Offspring viability indices:
Pre-implantation loss was calculated as a percentage from the formula:
(No. of corpora lutea – no. of implantations)/No. of corpora lutea x 100

Pre-birth loss was calculated as a percentage from the formula:
(No. of visible implantations – total litter size at birth )/No. of visible implantations x 100

Pup loss at birth was calculated as a percentage from the formula:
(Total litter size – live litter size)/Total litter size x 100

Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula:
(Total litter size at birth – live litter size at Day 4)/Total litter size at birth x 100

Sex ratios were calculated at birth and on Day 4 post partum and were presented as the percentage of males per litter.


Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY
No mortality occurred throughout the study.
No signs of toxicological significance were found. The majority of high dose animals showed slight to moderate staining of the tail throughout the
study. This finding was also recorded in the mid-dose group. No other clinical signs were recorded.
No signs were recorded in the low dose group throughout the study.
At observation of the cage tray, violet staining and/or dark faeces were recorded in the mid- and high dose groups during treatment.
These findings disappeared few days after the end of treatment during the recovery phase.

BODY WEIGHT AND BODY WEIGHT GAIN
No effects on body weight were found. A transient reduction in body weight gain was recorded in the high dose males of the main group after 2 weeksof treatment and at the end of the dosing period.

FOOD CONSUMPTION
Food consumption was unaffected by treatment. No differences were noted during the recovery period.

BLOOD CLOTTING TIME
No differences were noted in the in vivo coagulation performed at the end of the treatment phase in the main groups.

HAEMATOLOGY
No clear toxicological relevance was given to the neutrophilia and lymphopenia observed in some high dose males of the main group. Neutropenia
was instead observed in the high dose females. Comparable value of neutrophil was recorded in recovery animals. No change was recorded in the
coagulation test.

CLINICAL CHEMISTRY
Increased values of γ-glutamyltransferase and bilirubin were recorded in the mid- and high dose groups. At the end of recovery phase, almost
complete reversibility was noted.

URINALYSIS (main groups only)
No treatment-related changes were recorded.

ORGAN WEIGHTS
Some statistically significant differences were noted in the absolute or relative organ weights, such as:
Decrease in absolute thymus weight in males of the main groups receiving 1000 mg/kg bw/day (16.6%);
Increase in absolute thyroid weight in males of the recovery group receiving 1000 mg/kg bw/day (21.4%);
Increase in relative testes weight in males of the main groups receiving 1000 mg/kg bw/day (6%).
The above differences were not accompanied by histopathological findings. Therefore, they were considered not
treatment-related.

GROSS PATHOLOGY
Dark mucoid contents in the caecum and dark colour in the mesenteric lymph nodes, testes and kidneys were mainly reported in high dose males.
This renal change was also noted in most high dose females. In addition, dark staining was also reported in the tail.

HISTOPATHOLOGY
No treatment-related changes were reported.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Gross pathological findings:
no effects observed
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects/malformations were noted in offspring
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

OESTROUS CYCLE – BEFORE PAIRING PERIOD - GROUP SUMMARY DATA

STUDY NO.: X0010

-------------------------------------------------------------------------------------------------------------

          Group 1              Group 2             Group 3             Group 4

 Animal   Oestrus    Animal   Oestrus   Animal   Oestrus   Animal   Oestrus

 Number   Cycles     Number   Cycles    Number   Cycles    Number   Cycles

-------------------------------------------------------------------------------------------------------------

 X0010001    1      X0010021    1      X0010041    1      X0010061    2

 X0010003    0      X0010023    1      X0010043    1      X0010063    2

 X0010005    2      X0010025    1      X0010045    1      X0010065    1

 X0010007    1      X0010027    1      X0010047    1      X0010067    1

 X0010009    1      X0010029    1      X0010049    0      X0010069    1

 X0010011    2      X0010031    2      X0010051    2      X0010071    1

 X0010013    2      X0010033    1      X0010053    3      X0010073    2

 X0010015    2      X0010035    2      X0010055    0      X0010075    2

 X0010017    2      X0010037    2      X0010057    3      X0010077    2

 X0010019    2      X0010039    1      X0010059    2      X0010079    1

    Means  1.5                 1.3                 1.4                 1.5

-------------------------------------------------------------------------------------------------------------

 Note: The number of oestrus cycles is based on the number of non sequential days the dams were in oestrus.

REPRODUCTIVE PARAMETERS OF MALES–SUMMARY

------------------------------------------------------------------------------------

                             Group       1          2         3          4

------------------------------------------------------------------------------------

  Copulatory Index%            80.0      90.0       90.0      100.0

----------------------------------------------------------------------------------

  Fertility Index%                  70.0      70.0       80.0       90.0

------------------------------------------------------------------------------------

REPRODUCTIVE PARAMETERS OF FEMALES–SUMMARY

------------------------------------------------------------------------------------

                             Group       1          2         3          4

------------------------------------------------------------------------------------

  Copulatory Index%            100.0     100.0      100.0      100.0

------------------------------------------------------------------------------------

  Fertility Index%                   90.0      80.0       90.0       90.0

------------------------------------------------------------------------------------

Conclusions:
In conclusion, no toxic effects of Reactive Brown DYHY0331/0334 were seen after repeated dose and on reproduction/development in Sprague
Dawley rats. On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for both general toxicity and reproduction/developmental toxicity could be considered 1000 mg/kgbody weight/day for male and female rats of the parental generation and their offspring.
Executive summary:

Study design

A reproductive/developmental toxicity study, including evaluation of systemic toxicity after repeated dose of Reactive Brown DYHY 0331/0334, was conducted in Sprague Dawley SD rats up to Day 4 post partum. The animals received the test item, dissolved in softened water, at the dosages of 62.5, 250 and 1000 mg/kg body weight/day. Three groups of 10 males and 10 females each were administered orally by gavage with the test item at constant volume of 10 mL/kg body weight (Groups 2 to 4). A control group of the same number of animals/sex was administered with softened water only (Group 1). In addition, two groups (Groups 5 and 6) of five animals/sex were included and administered for four consecutive weeks at the same treatment conditions. A 4‑week treatment-free period was allowed for these two additional groups, in order to assess recovery from any delayed toxicity or persistence of adverse effects observed during the dosing phase.

Main groups

According to the study design, male animals were dosed 2 weeks prior to pairing, continuously thereafter during pairing and up to the day before necropsy (Week 7). Females were dosed throughout the study including 2 weeks before pairing, thereafter during pairing, gestation and lactation periods until Day 3 post partum.

The following parameters were evaluated in parental animals: body weight, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction to stimuli), food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology, clinical chemistry and urinalysis only males), litter data, macroscopic observations and organ weights. Histopathological examination was performed only on control and high dose groups (five animals/sex/group randomly selected). It included identification of the stages of the spermatogenic cycle in the selected five males. Measurements of body weight, clinical signs and macroscopic observations of pups were also performed.

Recovery groups

According to the study design, the animals were administered daily for 4 consecutive weeks followed by a 4‑week recovery period.

The following parameters were evaluated in these animals: body weight, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction to stimuli), food consumption, clinical pathology investigations (haematology and clinical chemistry), macroscopic observations and organ weights. As no adverse effects in the main groups were observed, no histopathology was performed.

Results

Mortality and fate of females

No mortality occurred throughout the study.

Non-pregnant females were found in all groups and specifically: one each in the control, mid- and high-dose groups and 2 in the low dose group.

Two females, one in the low dose group and one in the mid-dose group had unilateral implantation. The mid-dose female with unilateral implantation in the right horn had also total resorption in that horn and was not pregnant in the left one.

Therefore, the number of females with live pups on Day 4 post partum was: 9 in each of the control and high dose groups and 8 in each of the low and mid-dose groups.

Clinical signs, neurotoxicity assessment and observation of cage tray

Brown staining of the tail was recorded in the mid- and high dose groups. This finding was related to the colour of the test item (dark brown textile dye).

No signs were recorded in the low dose group throughout the study. Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item. In addition, measurements of motor activity, grip strength and sensory reaction to stimuli were comparable between control and treated groups.

At observation of the cage tray, violet staining and/or dark faeces were recorded in the mid- and high dose groups during treatment. These findings disappeared few days after the end of treatment during the recovery phase.

Body weight and body weight gain

No difference in body weights was recorded in animals of both sexes compared to the relative control groups (Groups 1 and 5), throughout the study. However, transient reduction in body weight gain was recorded in the high dose males of the main group after 2 weeks of treatment and at the end of the dosing period.

Food consumption

No effects on food consumption were observed in either males or females of all groups when compared to control groups.

Haematology

At the end of the treatment period, neutrophilia and lymphopenia were observed in some high dose males of the main group. On the contrary, neutropenia was observed in some high dose females. Due to the inconsistency between sexes, these changes were not conclusively attributed to treatment and therefore not considered of toxicological significance.

At the end of recovery period, neutrophilia showed almost complete recovery in males, whereas lymphocytes and neutrophils were still lower in treated males and females respectively, when compared to controls.

No changes of toxicological relevance were recorded in the coagulation tests (PT and APTT and blood clotting time).

Clinical chemistry

A dose-related increase of bilirubin was recorded in males and females of the main groups. Increased values of gamma-glutamyltransferase were also recorded, especially in the high dose group. These changes, especially for bilirubin, were expected (Sponsor’s communication) and likely due to the staining of the plasma by the test item (a dark brown textile dye), therefore interfering with the photometric determination of the parameters.

At the end of recovery phase, almost complete reversibility was noted.

Urinalysis

No treatment-related changes were recorded.

Oestrous cycle, reproductive parameters, pairing combination and mating performance

Oestrous cycle, pre-coital intervals, copulatory and fertility indices did not show intergroup differences.

Implantation, pre-birth loss data and gestation length of females

No significant differences were observed in the number of implantations, corpora lutea, total litter size, pre-implantation loss and pre-birth loss between control and treated groups. The majority of dams gave birth on Day 22 post coitum.

Litter data at birth, on Day 1 and on Day 4 post partum of females and sex ratio of pups

Reproductive outcomes of dams which included the number and body weight of pups and the percentage of live pups on Days 1 and 4 post partum did not differ between groups. Sex ratio of pups was also comparable between groups.

Clinical signs of pups

There were no test compound-related effects. Small pups, pups cold to touch and/or with apparently no food intake were generally observed in all groups including controls without substantial differences.

Necropsy findings in deceased pups and in pups sacrificed on Day 4 post partum

Necropsy findings in deceased pups and in pups sacrificed on Day 4 post partum did not reveal any treatment-related effect.

Terminal body weight and organ weights

Terminal body weight was unaffected by treatment in both sexes. No toxicological differences were seen in the absolute and/or relative organ weights between groups.

Macroscopic observations

The most relevant changes at macroscopic observations were dark mucoid contents in the caecum and/or dark colour in the mesenteric lymph nodes, testes and kidneys.

This colouration was attributed to the chemical properties of the test item, which is a dark brown textile dye.

Microscopic observations

No treatment-related changes were reported. In addition, no abnormalities were found at the evaluation of the spermatogenic cycle, regular layering in the germinal epithelium of seminiferous tubules was recorded.

Conclusion

In conclusion, no toxic effects of Reactive Brown DYHY0331/0334 were seen after repeated dose and on reproduction/development in Sprague Dawley rats. On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for both general toxicity and reproduction/developmental toxicity was considered to be 1000 mg/kg body weight/day for male and female rats of the parental generation and their offspring.

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Study design

A reproductive/developmental toxicity study, including evaluation of systemic toxicity after repeated dose of Reactive Brown DYHY 0331/0334, was conducted in Sprague Dawley SD rats up to Day 4 post partum. The animals received the test item, dissolved in softened water, at the dosages of 62.5, 250 and 1000 mg/kg body weight/day. Three groups of 10 males and 10 females each were administered orally by gavage with the test item at constant volume of 10 mL/kg body weight(Groups 2 to 4). A control group of the same number of animals/sex was administered with softened water only (Group 1). In addition, two groups (Groups 5 and 6) of five animals/sex were included and administered for four consecutive weeks at the same treatment conditions. A 4‑week treatment-free period was allowed for these two additional groups, in order to assess recovery from any delayed toxicity or persistence of adverse effects observed during the dosing phase.

Main groups

According to the study design, male animals were dosed 2 weeks prior to pairing, continuously thereafter during pairing and up to the day before necropsy (Week 7). Females were dosed throughout the study including 2 weeks before pairing, thereafter during pairing, gestation and lactation periods until Day 3post partum.

The following parameters were evaluated in parental animals: body weight, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction to stimuli), food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology, clinical chemistry and urinalysis only males), litter data, macroscopic observations and organ weights. Histopathological examination was performed only on control and high dose groups (five animals/sex/group randomly selected). It included identification of the stages of the spermatogenic cycle in the selected five males. Measurements of body weight, clinical signs and macroscopic observations of pups were also performed.

Recovery groups

According to the study design, the animals were administered daily for 4 consecutive weeks followed by a 4‑week recovery period.

The following parameters were evaluated in these animals: body weight, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction to stimuli), food consumption, clinical pathology investigations (haematology and clinical chemistry), macroscopic observations and organ weights. As no adverse effects in the main groups were observed, no histopathology was performed.

Results

Mortality and fate of females

No mortality occurred throughout the study.

Non-pregnant females were found in all groups and specifically: one each in the control, mid- and high-dose groups and 2 in the low dose group.

Two females, one in the low dose group and one in the mid-dose group had unilateral implantation. The mid-dose female with unilateral implantation in the right horn had also total resorption in that horn and was not pregnant in the left one.

Therefore, the number of females with live pups on Day 4post partum was: 9 in each of the control and high dose groups and 8 in each of the low and mid-dose groups.

Clinical signs, neurotoxicity assessment and observation of cage tray

Brown staining of the tail was recorded in the mid- and high dose groups. This finding was related to the colour of the test item (dark brown textile dye).

No signs were recorded in the low dose group throughout the study. Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item. In addition, measurements of motor activity, grip strength and sensory reaction to stimuli were comparable between control and treated groups.

At observation of the cage tray, violet staining and/or dark faeces were recorded in the mid- and high dose groups during treatment. These findings disappeared few days after the end of treatment during the recovery phase.

Body weight and body weight gain

No difference in body weights was recorded in animals of both sexes compared to the relative control groups (Groups 1 and 5), throughout the study. However, transient reduction in body weight gain was recorded in the high dose males of the main group after 2 weeks of treatment and at the end of the dosing period.

Food consumption

No effects on food consumption were observed in either males or females of all groups when compared to control groups.

Haematology

At the end of the treatment period, neutrophilia and lymphopenia were observed in some high dose males of the main group. On the contrary, neutropenia was observed in some high dose females. Due to the inconsistency between sexes, these changes were not conclusively attributed to treatment and therefore not considered of toxicological significance.

At the end of recovery period, neutrophilia showed almost complete recovery in males, whereas lymphocytes and neutrophils were still lower in treated males and females respectively, when compared to controls.

No changes of toxicological relevance were recorded in the coagulation tests (PT and APTT and blood clotting time).

Clinical chemistry

A dose-related increase of bilirubin was recorded in males and females of the main groups. Increased values of gamma-glutamyltransferase were also recorded, especially in the high dose group. These changes, especially for bilirubin, were expected (Sponsor’s communication) and likely due to the staining of the plasma by the test item (a dark brown textile dye), therefore interfering with the photometric determination of the parameters.

At the end of recovery phase, almost complete reversibility was noted.

Urinalysis

No treatment-related changes were recorded.

Oestrous cycle, reproductive parameters, pairing combination and mating performance

Oestrous cycle, pre-coital intervals, copulatory and fertility indices did not show intergroup differences.

Implantation, pre-birth loss data and gestation length of females

No significant differences were observed in the number of implantations, corpora lutea, total litter size, pre-implantation loss and pre-birth loss between control and treated groups. The majority of dams gave birth on Day 22post coitum.

Litter data at birth, on Day 1 and on Day 4 post partum of females and sex ratio of pups

Reproductive outcomes of dams which included the number and body weight of pups and the percentage of live pups on Days 1 and 4post partum did not differ between groups. Sex ratio of pups was also comparable between groups.

Clinical signs of pups

There were no test compound-related effects. Small pups, pups cold to touch and/or with apparently no food intake were generally observed in all groups including controls without substantial differences.

Necropsy findings in deceased pups and in pups sacrificed on Day 4 post partum

Necropsy findings in deceased pups and in pups sacrificed on Day 4post partum did not reveal any treatment-related effect.

Terminal body weight and organ weights

Terminal body weight was unaffected by treatment in both sexes. No toxicological differences were seen in the absolute and/or relative organ weights between groups.

Macroscopic observations

The most relevant changes at macroscopic observations were dark mucoid contents in the caecum and/or dark colour in the mesenteric lymph nodes, testes and kidneys.

This colouration was attributed to the chemical properties of the test item, which is a dark brown textile dye.

Microscopic observations

No treatment-related changes were reported. In addition, no abnormalities were found at the evaluation of the spermatogenic cycle, regular layering in the germinal epithelium of seminiferous tubules was recorded.

Conclusion

In conclusion, no toxic effects of Reactive Brown DYHY 0331/0334 were seen after repeated dose and on reproduction/development in Sprague Dawley rats. On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for both general toxicity and reproduction/developmental toxicity was considered to be 1000 mg/kg body weight/day for male and female rats of the parental generation and their offspring.

 


Short description of key information:
No adverse effects of Reactive Brown DYHY0331/0334 were seen after repeated dose and on reproduction/development in rats. On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for both general toxicity and reproduction/developmental toxicity was considered to be 1000 mg/kg body weight/day for male and female rats of the parental generation and their offspring.

Effects on developmental toxicity

Description of key information
No adverse effects of Reactive Brown DYHY0331/0334 were seen after repeated dose and on reproduction/development in rats. On the basis of the results obtained in the combined repeated dose/reproduction screening study, the NOAEL (No Observed Adverse Effect Level) for developmental toxicity was considered to be 1000 mg/kg body weight/day 
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
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
Justification for selection of Effect on developmental toxicity: via oral route:
No malformations were observed in the developmental screening study discussed above

Justification for classification or non-classification

Additional information