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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2011 - June 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:

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:

- Physical state/ appearance: solid / yellow
- Shape of particles: spherical
- Aspect ratio: 1.5
- Particle size distribution: 62.7 nm
- Crystal structure: crystalline
- Surface area of particles: 29.6 m^2/g
- Surface treatment: no

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

Details on exposure:

- applied as a suspension in water

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): pregnant animals and litter together

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- according to 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

Details on study schedule:

- Age at mating of the mated animals in the study: 13-14 weeks

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

Positive control:

no

Examinations

Parental animals: Observations and examinations:

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

Sperm parameters (parental animals):

stages of spermatogenesis were examined in histopathology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality up to day 4, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
yes, all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically

Postmortem examinations (parental animals):

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
ORGAN WEIGHTS: Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus

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

Postmortem examinations (offspring):

SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: All pups delivered from the F0 parents were examined as soon as possible on the day of birth

GROSS NECROPSY
All surviving pups (sacrificed on PND 4 under isoflurane anesthesia with CO2), all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically.

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

Reproductive indices:

Male reproduction data:
- Male mating index
- Male fertility index

Female reproduction and delivery data:
- Female mating index
- Female fertility index
- Gestation index
- Live birth index
- Post implantation loss

Offspring viability indices:

Pup number and status at delivery
Pup viability/mortality
Sex ratio
Pup body weight data

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, 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:

not examined

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-related.

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 doseresponse
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 doseresponse 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

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Histopathological examination of the stages of spermatogenesis did not reveal any adverse effects.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

- 2 control males, 2 male animals of test group 1, 3 male animals of test group 2 and 1 male animal of test group 3 did not generate F1 pups. Thus, the male fertility index ranged between 70% and 90%, what reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

- 1 control female, 2 females of test group 1, 3 females test group 2 and 1 female of test group 3 were either sperm-negative or did not become pregnant
- 1 control female and 2 females of test group 2 (300 mg/kg bw/d) were pregnant after mating but had not delivered viable pups (implantation sites only). Thus, the female fertility index varied between 80% and 90%. These findings were assessed to be incidental and not related to treatment as no dose-response relationship was observed.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Remarks on result:

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

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

- one pup of a female of test group 1 and one pup of a female of test group 3 (100 and 1000 mg/kg bw/d) were found dead. These findings were assessed to be incidental and not related to treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- Mean pup body weights were significantly lower on PND1 in test groups 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d).
- One female runt was seen in test group 2 (300 mg/kg bw/d) and 2 male and 3 female runts in test group 3 (1000 mg/kg bw/d).
These values were within the range of the biological variation inherent in the strain of rats used for this study.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- A situs inversus of the heart was observed in 1 pup of test group 3 (1000 mg/kg bw/d). The finding was assessed as being spontaneous in nature and without biological relevance.

Histopathological findings:

not examined

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Sex:

male/female

Remarks on result:

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

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1: Summary of female reproduction and delivery data

		TEST GROUP0 0 MG/KGBW/D	TEST GROUP 1 100 MG/KG BW/D	TEST GROUP 2 300 MG/KG BW/D	TEST GROUP 3 1000 MG/KG BW/D
Females on Study	N	10	10	10	10
Females Mated	N	l0Fi	10	8	10
Female Mating Index	%	100	100	80	100
Mating days until day 0 pc	MEAN	3.8 D	1.9	2.3	4.2
	S.D.	3.39	1.66	1.49	3.33
	N	10	10	8	10
days 1 to 4	N	9	9	8	9
	%	90	90	100	90
days 5 to 8	N	0	1	0	0
	%	0.0	10	0.0	0.0
days 9 to 14	N	1	0	0	1
	%	10	0.0	0.0	10
days 15 to 21	N	0	0	0	0
	%	0.0	0.0	0.0	0.0
Females Pregnant	N	9Fi	8	7	9
Female Fertility Index	%	90	80	88	90
Duration of Gestation (Days)	MEAN	22.1 D	22.0	21.8	21.9
	S.D.	0.35	0.00	0.45	0.33
Implantation sites	TOTAL	101	90	71	123
	MEAN	11.2 D	11.3	10.1	13.7
	S.D.	3.49	2.60	6.15	2.06
	N	9	8	7	9
Postimplantation Loss	TOTAL	5	1	3	2
	MEAN	0.6 D	0.1	0.4	0.2
	S.D.	0.88	0.35	0.79	0.44
	N	9	8	7	9
% Postimplantation Loss	MEAN	13.8 D	1.6	28.6	1.4
	S.D.	32.82	4.42	48.80	2.83
	N	9	8	7	9

Females withLiveborn	N	8Fi	8	5	9
Gestation Index	%	89	100	71	100
with Stillborn Pups	N	0Fi	1	0	0
	%	0.0	13	0.0	0.0
with all Stillborn	N	0Fi	0	0	0
	%	0.0	0.0	0.0	0.0
Pups Delivered	MEAN	12.0 D	11.1	13.6	13.4
	S.D.	0.93	2.80	2.07	1.81
	TOTAL	96	89	68	121
Liveborn	N	96Fi	88	68	121
Live Birth Index	%	100	99	100	100
Stillborn	N	0Fi	1	0	0
	%	0.0	1.1	0.0	0.0

Table 2: Summary of litter data

		TEST GROUP0 0 MG/KGBW/D	TEST GROUP 1 100 MG/KG BW/D	TEST GROUP 2 300 MG/KG BW/D	TEST GROUP 3 1000 MG/KG BW/D
(Total Number of) Litters	N	8	8	5	9
Litters with LivebornPups	N	8Fi	8	5	9
	%	100	100	100	100
Litters with StillbornPups	N	0Fi	1	0	0
	%	0.0	13	0.0	0.0
Litters with all Stillborn Pups	N	0Fi	0	0	0
	%	0.0	0.0	0.0	0.0
Pups Delivered	TOTAL	96	89	68	121
	MEAN	12.0 D	11.1	13.6	13.4
	S.D.	0.93	2.80	2.07	1.81
Pups Liveborn	N	96Fi	88	68	121
	%	100	99	100	100
Pups Stillborn	N	0Fi	1	0	0
	%	0.0	1.1	0.0	0.0
Pups Died	N	0Fi	1	0	1
	%	0.0	1.1	0.0	0.8
Pups Sacrificed Moribund	N	0Fi	0	0	0
	%	0.0	0.0	0.0	0.0
Pups Cannibalized	N	0Fi	0	0	0
	%	0.0	0.0	0.0	0.0
Pups Accidental Death	N	0	0	0	0
	%	0.0	0.0	0.0	0.0
Pups Sacrificed, Maternal Death	N	0	0	0	0
	%	0.0	0.0	0.0	0.0

		TEST GROUP0 0 MG/KGBW/D	TEST GROUP 1 100 MG/KG BW/D	TEST GROUP 2 300 MG/KG BW/D	TEST GROUP 3 1000 MG/KG BW/D
Pups dead day 0	N	0	0	0	0
	%	0.0	0.0	0.0	0.0
days 1 to 4	N	0	1	0	1
	%	0.0	1.1	0.0	0.8
Pups Surviving days O to 4	N	96Fi	87	68	120
Viability Index	%	100	99	100	99

Table 3: Summary of necropsy observations

		TEST GROUP0 0 MG/KGBW/D	TEST GROUP 1 100 MG/KG BW/D	TEST GROUP 2 300 MG/KG BW/D	TEST GROUP 3 1000 MG/KG BW/D
Litters Evaluated	N	8	8	5	9
Pups Evaluated	N	95	89	68	120
Live	N	95	88	68	120
Stillborn	N	0	1	0	0
HEART, SITUS INVERSUS
Pup Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	0.8
Litter Incidence	N	0Fi	0	0	1
	%	0.0	0.0	0.0	11
Affected Pups/Litter	MEAN%	0.0Wi	0.0	0.0	0.7
	S.D.	0.00	0.00	0.00	1.96

TOTAL PUP NECROPSY OBSERVATIONS
Pup Incidence	N	0	0	0	1
	%	0.0	0.0	0.0	0.8
Litter Incidence	N	0Fi	0	0	1
	%	0.0	0.0	0.0	11
Affected Pups/Litter	MEAN%	0.0Wi	0.0	0.0	0.7
	S.D.	0.00	0.00	0.00	1.96

Statistics: D=Dunnett-test (two-sided); Fi =Fisher's exact test (one-sided); Wi=Wilcoxon-test (one-sided)

* : p<=0.05 ** : p<=0.01

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. The NOAEL for reproductive performance, fertility and developmental toxicity was set to 1000 mg/kg bw/d in male and female Wistar rats.