Benzoic acid, 2,3,4,5-tetrachloro-6-cyano-, methyl ester, reaction products with 4-[2-(4-aminophenyl)diazenyl]-3-methylbenzenamine and methanol sodium salt (1:1)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

August 2011 - June 2012

Reliability:

2 (reliable with restrictions)

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:

3,4,5,6-tetrachloro-N-[2-(4,5,6,7-tetrachloro-2,3-dihydro-1,3-dioxo-1H-inden-2-yl)-8-quinolyl]phthalimide

EC Number:

250-063-5

EC Name:

3,4,5,6-tetrachloro-N-[2-(4,5,6,7-tetrachloro-2,3-dihydro-1,3-dioxo-1H-inden-2-yl)-8-quinolyl]phthalimide

Cas Number:

30125-47-4

Molecular formula:

C26H6Cl8N2O4

IUPAC Name:

4,5,6,7-tetrachloro-2-[2-(4,5,6,7-tetrachloro-1,3-dioxo-2,3-dihydro-1H-inden-2-yl)quinolin-8-yl]-1H-isoindole-1,3(2H)-dione

Test material form:

solid: nanoform

Details on test material:

solid yellow

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and ServiceGmbH, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks
- Weight at study initiation:
- Fasting period before study: no
- Housing: individually, following exceptions: During overnight matings, male and female mating partners were housed together. Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: August 9th, 2011 To:October 6th, 2011

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

suspension

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): water
- Storage temperature of food: RT

- applied as a suspension

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- according GLP
- stability of the test substance in drinking water for a period of 7 days at room temperature was proven before the start of the study
- method stability of test item in drinking water: UV/VIS spectroscopy

Duration of treatment / exposure:

The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males,
and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: range finder test 300 and 1000 mg/kg bw, no effects up to 1000 mg/kg bw
- Rationale for animal assignment (if not random): Randomization

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).

FOOD CONSUMPTION
- weekly
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 4

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the course of FOB
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of administration period
- Anaesthetic used for blood collection: Yes, anaesthetized using isoflurane (Isoba®, Essex GmbH, Munich, Germany)
- Animals fasted: Yes
- How many animals: 5/sex/dose

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of administration period
- Animals fasted: Yes
- How many animals: 5/sex/dose

URINALYSIS: Yes
- Time schedule for collection of urine: males: after mating, females: 1 day before end of administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: male: postnatal day 0, female: 10d after gestation
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
1. Adrenal glands
2. All gross lesions
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve
12. Esophagus
13. Extraorbital lacrimal gland
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Target organs
43. Testes (modified Davidson’s solution)
44. Thymus
45. Thyroid glands
46. Trachea
47. Urinary bladder
48. Uterus
49. Vagina

HISTOPATHOLOGY: Yes, control and high dose group, gross lesions in all animals
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymides
11. Heart
12. Ileum
13. Jejunum
14. Kidneys
15. Liver
16. Lung
17. Lymph nodes (mesenteric and axillary lymph nodes)
18. Ovaries
19. Oviducts
20. Peyer’s patches
21. Prostate
22. Rectum
23. Sciatic nerve
24. Seminal vesicles
25. Spinal cord (cervical, thoracic and lumbar cords)
26. Spleen
27. Stomach (forestomach and glandular stomach)
28. Testes
29. Thymus
30. Thyroid glands
31. Trachea
32. Urinary bladder
33. Uterus
34. Vagina

Statistics:

Blood parameters:
For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose
group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians
For parameters with unidirectional changes:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians

Urinalysis parameters: WILCOXON-test (one-sided)

Food consumption: DUNNETT-test (twosided)

fertility indices: FISHER'S EXACT test

Proportions of affected pups per litter with necropsy observations: WILCOXON-test

Weight parameters: KRUSKAL-WALLIS test

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- All male animals of test group 3 (1000 mg/kg bw/d) showed yellowish discolored feces towards the end of the study, i.e. on study days 33 and 34. Also all female animals of test group 3 (1000 mg/kg bw/d) showed yellowish discolored feces at the end of the study.
- In one female animal of test group 1 (100 mg/kg bw/d) abdominal distension was observed on study days 49 and 50.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female animal of test group 1 (100 mg/kg bw/d) was sacrificed moribund on study day 50 (the animal was not pregnant). It revealed dilation of uterus and cervix with a cloudy fluid content. This was histopathologically diagnosed as a diffuse inflammation with dilation and was regarded to be the cause for the infertility of this mating pair and also the reason for sacrificing this animal. It was a single case and not regarded to be treatment-related but spontaneous in origin.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- During the first premating week as well as in the first gestation week the food consumption in female animals of test group 3 (1000 mg/kg bw/d) was significantly increased. These findings were assessed as spontaneous in nature and not test substance-related.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- In males of test groups 1 and 2 (100 and 300 mg/kg bw/d), creatinine values were lower compared to controls, but the means were not dose-dependently decreased. Therefore, this alteration was regarded as incidental and not treatment-relat

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Functional observational battery: Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test substance-treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental.
- Motor activity measurement: Deviations to the control were only noted in test group 1 (100 mg/kg bw/d), i.e. increased value in interval 9 in male animals and increased value in interval 6 in female animals. As no significant deviations were noted with regard to the overall motor activity and no dose-response relationship was observed, the findings were assessed as being incidental.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- The weight increase in absolute and relative spleen weight in females and in relative spleen weight in males of test group 2 (300 mg/kg bw/d) was regarded to be incidental due to a missing dose-response relationship and missing histopathologic findings in test group 3 (1000 mg/kg bw/d).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- 2 males and 7 females of test group 3 (1000 mg/kg bw/d) revealed a yellow discoloration of the glandular stomach contents. 3 females of test group 3 (1000 mg/kg bw/d) showed the same discoloration of the contents of the jejunum.
- One female animal of test group 1 (100 mg/kg bw/d) revealed a yellow discoloration of the lung (regarded to be test substance that was aspired subsequently to the gavage procedure or due to a gavage error into the trachea) and the mediastinal lymph nodes (regarded to be the physiologic clearing route of the lung).

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 2 females of test group 3 (1000 mg/kg bw/d) revealed histopathological yellow crystalline particles within the alveoli, often close to or within macrophages, rarely inside multinucleated giant cells. The same finding was also observed for the female animal of test group 1 that revealed the macropscopic finding “discoloration” in the lung. These yellow particles were regarded to be test substance that was aspired subsequently to the gavage procedure. Therefore, these discolorations in lung and mediastinal lymph node were caused by the test substance but were not regarded to be a treatment-related adverse finding.

Histopathological findings: neoplastic:

no effects observed

Other effects:

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:

other: no adverse effects observed up to and including the limit dose

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration by gavage to male and female Wistar rats did not reveal signs of toxicity. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d in both sexes.

Executive summary:

The test substance was administered orally by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0, drinking water served as vehicle), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females.

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear. A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4. Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating and mating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, day of parturition (postnatal day [PND] 0) and on PND 4.

Clinicochemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group towards the end of the administration period. Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 animals per sex and test group.

All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration by gavage to male and female Wistar rats did not reveal signs of toxicity. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d in both sexes.