Bis(2-chloroethyl) 3,3'-[(2,5-dimethyl-p-phenylene)bis[iminocarbonyl(2-hydroxy-1,3-naphthylene)azo]]di-p-toluate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-chloroethyl) 3,3'-[(2,5-dimethyl-p-phenylene)bis[iminocarbonyl(2-hydroxy-1,3-naphthylene)azo]]di-p-toluate

EC Number:

269-507-4

EC Name:

Bis(2-chloroethyl) 3,3'-[(2,5-dimethyl-p-phenylene)bis[iminocarbonyl(2-hydroxy-1,3-naphthylene)azo]]di-p-toluate

Cas Number:

68259-05-2

Molecular formula:

C50 H42 Cl2 N6 O8

IUPAC Name:

2-chloroethyl 2-[(2Z)-2-[3-[[4-[[(4E)-4-[[5-(2-chloroethoxycarbonyl)-2-methylphenyl]hydrazinylidene]-3-oxonaphthalene-2-carbonyl]amino]-2,5-dimethylphenyl]carbamoyl]-2-oxonaphthalen-1-ylidene]hydrazinyl]-3-methylbenzoate

Test material form:

solid: particulate/powder

Details on test material:

CAS No. 68259-05-2
Description: Red powder
Purity: 99.55 wt%
Expiry Date (Retest Date): 18-Jul-2020
Storage Conditions: Room temperature (20 ± 5 °C)

Specific details on test material used for the study:

Description: Red powder
Purity: 99.55 wt%
Expiry Date (Retest Date): 18-Jul-2020
Storage Conditions: Room temperature (20 ± 5 °C)

Test animals

Species:

rat

Strain:

other: RccHanTM: WIST(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., 5961 NM Horst / Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 315 to 364 g, Females: 182 to 212 g
- Fasting period before study: none
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2011-12-15 To: 2012-02-06

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared fresh daily using the test item as supplied by the Sponsor.


VEHICLE
- Concentration in vehicle: as adjusted to dose
- Amount of vehicle (if gavage): 10 mL/kg body weight
:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day samples from the control group as well as three samples (top, middle
and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of
concentration and homogeneity. During the last week of the treatment, samples were taken from
the middle to confirm concentration.

Duration of treatment / exposure:

Males: Minimum 4 weeks
Females: Approximately 6 weeks

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Experimental data on substance of similar structure

Positive control:

Not required

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (twic for mortality)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first administration of the test item and weekly thereafter (in the gestation period on day 0, 6, 13 and 20 post coitum), detailed clinical observations were performed outside the home cage in a standard arena.
- Cage side observations : changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g.
lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.


BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data



OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained on the day before or on the day of the scheduled necropsy from
5 males randomly selected from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia.)
- Animals fasted: Yes
- How many animals: 5
- Parameters checked below were examined:

Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Leukocyte count, total
Differential leukocyte count
Platelet count
Reticulocytes
Prothrombin time (= Thromboplastin time)
Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were obtained on the day before or on the day of the scheduled necropsy from
5 males randomly selected from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum
- Animals fasted: Yes
- How many animals: 5
- Parameters checked below were examined:

Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Aspartate aminotransferase
Alanine aminotransferase
Alkaline phosphatase
Gamma-glutamyl-transferase
Bile acids
Sodium
Potassium
Chloride
Calcium
Phosphorus
Protein, total
Albumin
Globulin
Albumin/Globulin ratio

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum) relevant parameters were performed with five P generation males and five P generation females randomly selected from each group. This FOB assessment was conducted following the daily dose administration.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)

At the scheduled sacrifice, the testes and epididymides of 5 parental males were weighed separately.

-Organ weights (5 animals):
Adrenal glands (weighed as pairs)
Brain
Heart
Kidneys (weighed as pairs)
Uterus (including cervix)
Prostate
Liver
Thymus
Spleen
Thyroid (after fixation)
Ovaries (weighed as pairs)
Seminal vesicles (inclusive coagulating gland)


HISTOPATHOLOGY: Yes
Testes, epididymides, prostate, seminal vesicles, ovaries, oviduct, vagina and uterus from all animals of the control and high-dose group were examined. The remaining organs/tissues of 5 randomly selected males and females of the control and high-dose group, respectively, were examined histopathologically:

Brain
Spinal chord (cervical, thoracic, lumbar)
Small and large intestines (duodenum, jejunum, ileum, colon, caecum, rectum, incl. Peyer’s patches)
Stomach (forestomach and glandular stomach)
Liver
Kidneys
Adrenals
Lymph nodes (axillary and mesenteric)
Urinary bladder
Aorta1
Eyes with optic nerve and harderian gland1
Lacrimal gland1
Larynx1
Nasal cavity 1
Esophagus1
Heart
Thymus
Thyroids and parathyroids
Trachea and lungs (preserved by inflation
with fixative and then immersion)
Pituitary gland1
Spleen
Peripheral nerve (sciatic)
Bone marrow (femur)
Femur with knee joint1
Mammary gland (male and female) 1
Pancreas1
Salivary glands – mandibular, sublingual 1
Skeletal muscle 1
Sternum with bone marrow1
Pharynx1
1 = only examined by histopathology in case of macroscopic findings indicative of potential toxicity

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:
• Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Effect levels

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

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

All males and females had reddish discolored feces one day after treatment start onwards until the end of the treatment period. The severity of this finding was dose-related. This finding is considered to be a typical effect resulting from oral administration of a red dyestuff and is without toxicological relevance.

Applicant's summary and conclusion

Conclusions:

The substance does not cause adverse effects upon 28-day gavage application to rats at the limit dose of 1000 mg/kg bw.