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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 90 days. Animals of an additional control group were handled identically as the dose groups but received 2 % Chremophor in aqua ad injectionem, the vehicle used in this study. The 4 groups comprised of 10 male and 10 female Wistar rats.
The following doses were evaluated:
Control: 0 mg/kg body weight
Low Dose: 250 mg/kg body weight
Medium Dose: 500 mg/kg body weight
High Dose: 1000 mg/kg body weight
The test item formulation was prepared within 12 days. The test item was dissolved in 2 % Chremophor in aqua ad injectionem and administered daily during a 90-day treatment period to male and female animals. Dose volumes were adjusted individually based on weekly body weight measurements.
During the period of administration, the animals were observed precisely each day for signs of toxicity. Animals that died were examined macroscopically and histopathologically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically and histopathologically. To evaluate possible toxic effects on fertility, the estrous cycle was examined at the end of the treatment period and epididymal sperm motility, testicular sperm count and sperm morphology were evaluated at the end of the treatment period.
A battery of functional observation tests was performed to detect possible neurotoxic effects of the test item.
Body weight and food consumption were measured weekly. At the conclusion of the treatment period, all animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved.
A full histopathological evaluation of the tissues was performed on high dose and control animals at the end of the treatment period and any animal found dead or euthanised before the planned day of sacrifice. For testis, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The test is performed in the rat. The rat is a widely used rodent species in toxicological studies and acceptable to regular authorities.
Sex:
male/female
Details on test animals and environmental conditions:
Test System
Species/strain: Healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of
the treatment period: approx. 7-8 weeks old
Body weight at the
allocation of the
animals to the
experimental groups: males: 175 – 208 g
(mean: 188.43 g, ± 20 % = 150.74 – 226.11 g)
females: 125 – 149 g
(mean: 135.23 g, ± 20 % = 108.18 – 162.27 g)

The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups of 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days)

Number and Sex of the Animals
80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item formulation or vehicle was administered at a single dose to the animals by oral gavage. The application volume for all groups was 10 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Vehicle:
other: The vehicle was prepared by mixing the required amount of Chremophor and aqua ad injectionem to receive 2 % Chremophor in aqua ad injectionem.
Details on oral exposure:
Characterisation of the Vehicle
The vehicle used in this study was selected based on the test item’s characteristics. The vehicle to be used in this study was 2 % Chremophor in aqua ad injectionem (sterile water).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration analysis of formulation samples was determined at three concentrations, 25 mg/mL, 50 mg/mL and 100 mg/mL in study weeks 1, 5, 9 and in the last week of the study. The mean recoveries observed for the LD dose group was between 78.9 % and 88.2 % of the nominal value, between 80.9% and 95.8% for the MD dose group and between 87.6 % and 111.0 % of the nominal value for HD dose group. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 83.5 %, 86.8 %, and 98.2 % of the nominal concentration, respectively.
Nominal concentrations were confirmed for MD and HD dose groups, as measured concentrations were within acceptance criterion of 15%. LD samples mean recovery was too low and out of acceptance criteria. All runs were valid in analytical point of view, as run QC samples were within acceptance criteria.
Homogeneity of formulation samples was determined at three concentrations, 25 mg/mL, 50 mg/mL and 100 mg/mL, in study weeks 1, 5, 9 and the last week of the study.
The coefficients of variation of the different sampling locations (top, middle, bottom) was between 1.6 % and 3.6 % in LD dose group, between 0.3 % and 13.8 % in MD dose group and between 1.2 % and 9.4 % in HD dose group. All samples were homogenous, as COV was below or equal 15 %.
Duration of treatment / exposure:
90 days.
Frequency of treatment:
Once daily.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (Group 1).
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Group 2.
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Group 3.
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4.
No. of animals per sex per dose:
80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).
Control animals:
yes, concurrent vehicle
Details on study design:
According to the results of a previous dose range finding study (BSL Munich Study No. 180632, non GLP) and in consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose = 250 mg/kg/day, MD = medium dose = 500 mg/kg/day, HD = high dose = 1000 mg/kg/day). The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 90 days.
Positive control:
None.

Examinations

Observations and examinations performed and frequency:
Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period.
Food consumption was measured weekly during the treatment period.

Clinical Observations
All animals were observed for clinical signs during the entire treatment period of 90 days. Inadvertently clinical observations were not performed on study day 1 and 36 for animals nos. 1-5 (control group, male), 11-15 (LD group, male), 21-25 (MD group, male) and 31-35 (HD group, male), on study day 35 for animals nos. 6-10 (control group, male), 16-20 (LD group, male), 26-30 (MD group, male) and 36-40 (HD group, male), on study day 34 for animals nos. 41-45 (C group, female), 51-56 (LD group, female), 61-65 (MD group, female) and 71-75 (HD group, female) and on study day 33 for animals nos. 46-50 (control group, female), 56-60 (LD group, female), 66-70 (MD group, female) and 77-80 (HD group, female).
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.
Ophthalmological examination, using an ophthalmoscope was made on all animals before the first administration and in the last week of the treatment period.

Functional Observations
Once before the first exposure and once in the last week of exposure multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests. These tests were conducted in all animals with exception of female animal nos. 76-80 in the HD group. Observations for these animals were not recorded in week 13.

Examination of Fertility Parameters
Daily over a period of 8 days, the estrous cycle of all female animals was examined in the last week of treatment.
At necropsy (one day after the last administration) left epididymis and left testis were separated and used for evaluation of sperm parameters. Epididymal sperm motility and testicular sperm count were evaluated in all male animals using Hamilton Thorne Sperm Analyser (TOX IVOS Version 13.0).

Haematology
Haematological parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.
The following haematological parameters were examined:
haematocrit value (HCT)
haemoglobin content (HGB)
red blood cell count (RBC)
mean corpuscular volume (MCV)
mean corpuscular haemoglobin (MCH)
mean corpuscular haemoglobin concentration (MCHC)
reticulocytes (Ret)
platelet count (PLT)
white blood cells (WBC)
neutrophils (Neut)
lymphocytes (Lym)
monocytes (Mono)
eosinophils (Eos)
basophils (Baso)
large unstained cells (Luc)

Blood Coagulation
Coagulation parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in citrate tubes.
The following coagulation parameters were examined:
prothrombin time (PT)
activated partial thromboplastin time (aPTT)

Clinical Biochemistry
Parameters of clinical biochemistry were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes.
The following parameters of clinical biochemistry were examined:
alanine aminotransferase (ALAT)
aspartate-aminotransferase (ASAT)
alkaline phosphatase (AP)
creatinine (Crea)
total protein (TP)
albumin (Alb)
urea
total bilirubin (TBIL)
total bile acids (TBA)
total cholesterol (Chol)
low density lipoprotein (LDL)
high density lipoprotein (HDL)
glucose (Gluc)
sodium (Na)
potassium (K)

For an evaluation of test item-related effects on the pituitary-thyroid axis and thyroid hormones, serum samples of all animals will be retained at the end of treatment (80 animals) and stored at < -15° C. T3, T4, TSH, testosterone, FSH and estradiol serum levels will be determined of all study animals (80 animals):
T3
T4
TSH
testosterone
FSH
estradiol

Urinalysis
A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance were recorded.
The following parameters were measured using qualitative indicators (Henry Schein Urine Stripes URI 10SL):
specific gravity
nitrite
pH-value (pH)
protein
glucose
ketone bodies (Ket)
urobilinogen (UBG)
bilirubin (BIL)
erythroctes (Ery)
leukocytes (Leuc)






Sacrifice and pathology:
Pathology
Gross necropsy
One day after the last administration (study day 91) all surviving animals of the treatment period were sacrificed using anesthesia (ketamine/ xylazin) and were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Organ Weight
The wet weight of following organs was taken from sacrificed animals as soon as possible. Paired organs were weighed together. Organ weights of animals found dead or euthanised for animal welfare reasons were not taken. Weight of thyroid/parathyroid glands was measured after fixation.
Tissue/Organ
liver
kidneys
adrenals
testes
epididymides
prostate, seminal vesicles and
coagulating glands
ovaries
uterus with cervix
thymus
thyroid/ parathyroid glands*
spleen
brain
pituitary gland
heart

The following tissues from all animals were preserved gender specific in 4 % neutral-buffered formaldehyde except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70 % ethanol.
adrenal glands
all gross lesions
aorta
brain (incl. medulla/pons, cerebellar
and cerebral cortex)
caecum
colon
duodenum
epididymides (right side)
heart
ileum (including Peyer´s patches)
jejunum
kidneys
liver
lungs
lymph nodes (mesenteric and axillary)
mammary gland area (male and female)
oesophagus
ovaries
pancreas
pituitary
prostate and seminal vesicles with coagulating glands as a whole
rectum
salivary glands (sublingual, submandibular, parotis)
sciatic nerve
skeletal muscle
skin
spinal cord (cervical, thoracic and lumbar segments)
spleen
sternum (with bone marrow)
stomach
testes (right side)
thymus
thyroid gland including parathyroid glands
trachea
urinary bladder
uterus with cervix and vagina

Histopathology
The afore-listed organs were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining for the animals of the groups 1 and 4 sacrificed at the end of the treatment period and any animal found dead or euthanised before the planned day of sacrifice. For testis, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
These examinations were not extended to animals of all other dosage groups for treatment-related changes as there were no test item-related findings observed in the high dose group.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.





Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with Ascentos 1.3.4 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Regular finding at clinical observation was discoloured faeces in the HD, MD and LD group animals during the observation period. Other findings like slightly reduced spontaneous activity and crust in the MD group, abnormal breathing in the HD group and diarrhoea in MD and HD animals were noted occasionally in single animals during the treatment period. As these findings in the dose groups were seen in single animals, not consistent over time and/or not consistent over dose groups, a test item-related effect is not considered and they are therefore considered to be incidental.
Occasional observations of moving the bedding and moderate salivation, seen for single LD and/or HD animals on several days were observed in timely relation to the dose administration and therefore considered to be signs of a local reaction after gavage manipulation rather than a systemic adverse effect.
At detailed clinical observation, a statistical significant increase of 20% above the mean value of the control group was observed for the group mean value of the parameter salivation in the female HD group in week 2. This finding in one single week is considered to be incidental.
Mortality:
no mortality observed
Description (incidence):
Male HD animal no. 35 was euthanized on study day 24 in moribund conditions. In females, mortalities were seen in LD and MD group. MD animal no. 61 and LD animal no. 52 were euthanized on study day 6 and 76. Animals no. 62 (MD female) and animal no. 54 (LD group) were found dead in cage on day 2 and 76.
Histopathologically, the cause of morbidity was considered to be related to the presence of blue fluid (test item) in the thoracic cavity, in the trachea or in the nasal cavities. Considering the results of histopathological assessment the cause of morbidity in the above-mentioned animals was related to the gavage procedure and is not considered a test item related effect and, as a consequence, was deemed to be incidental.
All remaining animals survived until the end of the treatment period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In the first week of treatment, statistical significance was found for a moderate decrease in body weight change in the male HD group (body weight increase days 1 to 8: 35.5 (controls); 24.6 (HD males). No further statistical significances were noted for body weight and body weight change in male and female dose groups.
All other mean values were statistically comparable to control groups. For the male HD group, body weight change from day 1 to 90 was not statistically but slightly decreased when compared to control group; body weight increase days 1 to 90: 181.2 (controls); 151.9 (HD males).
In males and females of all groups, there was an increase of body weight with the progress of the study, therefore the reduced body weight gain in the first week of treatment is considered to be incidental and no adverse test item-related effect on body weight development is considered.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no test item-related effect on food consumption in any of the dose groups. During the overall treatment period, the mean daily food consumption was comparable between all male and female dose groups and controls.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Statistical significant changes in haematological and coagulation parameters were seen in the male HD and MD groups, but not in females.
Regarding haematology, a very slight statistical significant increase was noted for MCHC in the male HD group (2.18 % above control) and for a slight decrease of mean PLT in the male LD and HD group (LD: 10.3% below control, HD: 10.1 % below control). Mean PT was slightly and statistically significantly decreased in the male MD and HD group (MD: 9.00 % below control, HD: 9.18 % below control). All other mean values in the male and female dose groups, were comparable to control group and no specific finding was noted.
As the statistical differences were slight, seen only in males and mean values were within historical control data range, a toxicological relevance is not considered.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Few parameters with statistical significances were observed in the male HD group when compared to control group.
Mean value for ALAT, ASAT, Crea, TP and ALB were slightly and statistically significantly decreased in the male HD group (ALAT: 20.13 % below control, ASAT: 22.54 % below control, Crea: 24.8 % below control, TP: 6.74 % below control, ALB: 6.553 % below control). No parameters with statistical significance were seen in all female dose groups. As the statistical differences were slight and mean values were within historical control data range, a toxicological relevance is not considered. Additionally, there were no histopathological findings related to an effect of the test item.
Hormone analysis showed no statistical significance for T3, T4, TSH, FSH, estradiol and testosterone in the male and female dose groups when compared to control group. No test item-related effect of treatment with test item was considered.
Urinalysis findings:
no effects observed
Description (incidence and severity):
The urinalysis of animals sacrificed at the end of the treatment period revealed no test item-related effects.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
In the week before start of treatment with test item, statistical significance was found in the male LD group for a decrease of animal sleeps and increased moving in cage. For females, slight statistical significant increase of animal sleeps and decrease of moving in cage was seen in the week before start of treatment in the HD animals.
In week 13 male mean score of animal sleeps was statistically significantly decreased (55.6% below control) and mean score of moving in cage slightly and statistically significantly increased in the male HD group.
The single observation in the male HD group are not considered to be of toxicological relevance as no other functional observations are observed in males and no such findings were seen in female groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weights with statistical significance were seen for male but not for female dose groups.
Final mean body weight at necropsy was slightly and statistically significant decreased for the male HD group (10.3 % below control) and additionally, a slight statistical significant decrease was found for mean organ weight of heart to brain (10.6 % below control). Mean organ weight to body weight for kidneys showed a slight statistical significant increase (8.1 % above control) when compared to control.
As there were no test item-related findings at histopathological evaluation and statistical significances were only slight and additionally not observed in female groups, the significances are not considered to be of toxicological relevance and are deemed to be incidental.
Other absolute and relative mean organ weights are comparable between the control and dose groups and no relevant differences were seen. No toxicological effect of the test item is therefore considered. In absence of corresponding microscopic changes all observed body and organ weight changes were considered of no toxicological relevance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The observed gross lesions at necropsy were deemed to be incidental, and considered not to be induced the test item. Further, all blue discolorations observed at necropsy in several organs were related to the colour of the test item (Direct Blue 264) and were considered to be of no pathological significance.
In absence of corresponding microscopic changes all observed body and organ weight changes were considered of no toxicological relevance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Regarding microscopic evaluation, all recorded findings in survivors, moribund and decedent animals were deemed to be incidental or were within the range of background alterations that may be recorded in Wistar rats.
At histopathological evaluation, the testes were checked on completeness of cell populations, completeness of stages and degenerative changes. No treatment-related effects on the testicular histomorphology were observed. Further, no treatment-related effect on interstitial cell structure was noticed.
There were neither macroscopic lesions nor histological changes in this study that could be attributed to the treatment with the test item. Thus the histopathological NOEL (no observed effect level) may be established at 1000 mg/kg bw.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: 1000 mg/kg/day was the highest applied dose.

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

On the basis of the present OECD TG 408 study, the 90-Day Repeated Dose Oral Toxicity study with Direct Blue 264 in male and female Wistar rats, with dose levels of 250, 500, and 1000 mg/kg body weight day the following conclusions can be made:

No test-item related mortality was observed and no adverse effects of the test item were found for male and female clinical observations, functional observations, body weight development, food consumption, hormone analysis, sperm parameters, haematology and coagulation, clinical biochemistry, urinalysis, gross macroscopic findings at necropsy, organ weights and histopathology in all treated dose groups.

The no observed adverse effect level (NOAEL) of Direct Blue 264 in this study is considered to be 1000 mg/kg body weight/day.

Applicant's summary and conclusion

Conclusions:
On the basis of the present OECD TG 408 study, the 90-Day Repeated Dose Oral Toxicity study with Direct Blue 264 in male and female Wistar rats, with dose levels of 250, 500, and 1000 mg/kg body weight day the following conclusions can be made:
No test-item related mortality was observed and no adverse effects of the test item were found for male and female clinical observations, functional observations, body weight development, food consumption, hormone analysis, sperm parameters, haematology and coagulation, clinical biochemistry, urinalysis, gross macroscopic findings at necropsy, organ weights and histopathology in all treated dose groups.
The no observed adverse effect level (NOAEL) of Direct Blue 264 in this study is considered to be 1000 mg/kg body weight/day.
Executive summary:

The test item Direct Blue 264 (CAS 68411-04-1) was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 90 days. Animals of an additional control group were handled identically as the dose groups but received 2 % Chremophor in aqua ad injectionem, the vehicle used in this study. The 4 groups comprised of 10 male and 10 female Wistar rats.

The following doses were evaluated: 0 (control), 250, 500, or 1000 mg/kg body weight.

The test item formulation was prepared within 12 days. The test item was dissolved in  2 % Chremophor in aqua ad injectionem and administered daily during a 90-day treatment period to male and female animals. Dose volumes were adjusted individually based on weekly body weight measurements.

During the period of administration, the animals were observed precisely each day for signs of toxicity. Animals that died were examined macroscopically and histopathologically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically and histopathologically. To evaluate possible toxic effects on fertility, the estrous cycle was examined at the end of the treatment period and epididymal sperm motility, testicular sperm count and sperm morphology were evaluated at the end of the treatment period.

A battery of functional observation tests was performed to detect possible neurotoxic effects of the test item.

Body weight and food consumption were measured weekly. At the conclusion of the treatment period, all animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved.

A full histopathological evaluation of the tissues was performed on high dose and control animals at the end of the treatment period and any animal found dead or euthanised before the planned day of sacrifice. For testis, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.

On the basis of the present OECD TG 408 study, the 90-Day Repeated Dose Oral Toxicity study with Direct Blue 264 in male and female Wistar rats, with dose levels of 250, 500, and 1000 mg/kg body weight day the following conclusions can be made:

No test-item related mortality was observed and no adverse effects of the test item were found for male and female clinical observations, functional observations, body weight development, food consumption, hormone analysis, sperm parameters, haematology and coagulation, clinical biochemistry, urinalysis, gross macroscopic findings at necropsy, organ weights and histopathology in all treated dose groups.

The no observed adverse effect level (NOAEL) of Direct Blue 264 in this study is considered to be 1000 mg/kg body weight/day.