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)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity after repeated dose administration of the test item was not evaluated; reliable, experimental data of an analogue are available. Reproductive toxicity of the analogue was determined in the course of a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening study (OECD 422, GLP). Oral administration by gavage to male and female Wistar rats did not reveal signs of toxicity. The NOAEL for reproductive performance, fertility and developmental toxicity was set to 1000 mg/kg bw/d in male and female Wistar rats.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

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

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

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

Route of administration:

oral: gavage

Vehicle:

water

Details on 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 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 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

Dose / conc.:

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

Dose / conc.:

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

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

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

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: 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.

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

no

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

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.

Executive summary:

A study according to OECD guideline 422 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. The NOAEL for reproductive performance, fertility and developmental toxicity was set to 1000 mg/kg bw/d in male and female Wistar rats.

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

Read across justification

Toxicity after repeated dose administration of the test item was not evaluated; reliable, experimental data of an analogue are available.

The substances share high similaritiy in structure and have comparable physico-chemical properties. Both substances are solids of poor water solubility and insoluble in most of the common organic solvents. The molecular weight of both compounds is higher than 600 g/mol. The molecules includes phthalimid-like structures and bear the potential to release chlorinated phthalimid after enzymatic or bacterial cleavage. Therefore, the analogue substance was choosen to examine toxicity after repeated dose administration and toxicity to reproduction and development.

Procedure and observations

The test item (analogue) 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 (drinking water served as vehicle), 100 mg/kg bw/d, 300 mg/kg bw/d and 1000 mg/kg bw/d. 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. A detailed clinical observation was performed in all animals. Body weights and food consumption were determined in F0 animals. Clinicochemical and hematological examinations as well as urinalyses and FOB were performed in all animals towards the end of the administration period. All animals were assessed by gross pathology; weights of selected organs were recorded and a histopathological examination was performed. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed with CO2 and examined macroscopically for external and visceral findings.

Clinical examinations, reproductive performance, clinical pathology, histopathology and gross pathology did not reveal any finding in treated animals. Clinical examination and gross pathology did not reveal any effect on pups.

Discussion

Thus, 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.

Effects on developmental toxicity

Description of key information

Toxicity after repeated dose administration of the test item was not evaluated; reliable, experimental data of an analogue are available. Reproductive toxicity of the analogue was determined in the course of a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening study (OECD 422, GLP). Oral administration by gavage to male and female Wistar rats did not reveal signs of toxicity. The NOAEL for reproductive performance, fertility and developmental toxicity was set to 1000 mg/kg bw/d in male and female Wistar rats.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

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

Qualifier:

according to guideline

Guideline:

other: OECD 422

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

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

Route of administration:

oral: gavage

Vehicle:

water

Details on 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

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight for maximum 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

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

Dose / conc.:

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

Dose / conc.:

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

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

Maternal 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

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

- External examinations: Yes, all pubs
- Soft tissue examinations: necropsy observations macroscopically
- Skeletal examinations: No
- Head examinations: No
- Sex ratio
- Pup body weight data
- Pup clinical observations

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

Indices:

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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- 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 of the low dose group (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:

no effects observed

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 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 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):

- 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).
These discolorations were caused by the test substance but were not regarded to be a treatmentrelated adverse finding.

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

Number of abortions:

not examined

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The mean postimplantation loss was highest in test group 2 (300 mg/kg bw/d), i.e. 28.6% compared to the control group (13.8%). As no dose-response relationship was observed, the finding was assessed as not being related to treatment.

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

not examined

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

1 female of the control group, 2 females of test group 1 (100 mg/kg bw/d), 3 females of test group 2 (300 mg/kg bw/d) and 1 female of test group 3 (1000 mg/kg bw/d) were either sperm-negative or did not become pregnant. These findings were assessed to be incidental and not related to treatment as no dose-response relationship was observed.

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

(instead of fetal body weight changes, the body weight of the pups was assessed)

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.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

one stillborn pup in test group 1 (100 mg/kg bw/d)

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

The viability index as indicator for pup mortality between PND 0 and 4 was 99% for test groups 1 and 3 (100 and 1000 mg/kg bw/d; 1 pup of 1 female in group 1 and 3 was found dead).
These findings were assessed to be incidental and not related to treatment.

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

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

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

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

Abnormalities:

not examined

Developmental effects observed:

no

Table 1: Summary of female reproduction and delivery data

		ORAL ADMINISTRATION(GAVAGE) - F0 FEMALES (Fl LITTER) SUMMARY OF FEMALE REPRODUCTION AND DELIVERY DATA
		TEST GROUP 0 0 MG/KG BW/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	10Fi	10	8	10
Female Mating Index	%	100	100	80	100
Mating days until day 0 pc	MEAN	3.8D	1.9	2.3	4.2
	S.D.	3.39	1.66	1.49	3.33
	N	10	10	8	10
days 1 to4	N	9	9	8	9
	%	90	90	100	90
days 5 to8	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	22.1D	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

		TEST GROUP 0 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 with Liveborn	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

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

Executive summary:

A study according to OECD Guideline 422 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. The NOAEL for reproductive performance, fertility and developmental toxicity was set to 1000 mg/kg bw/d in male and female Wistar rats.

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

Read across justification

Toxicity after repeated dose administration of the test item was not evaluated; reliable, experimental data of an analogue are available.

The substances share high similaritiy in structure and have comparable physico-chemical properties. Both substances are solids of poor water solubility and insoluble in most of the common organic solvents. The molecular weight of both compounds is higher than 600 g/mol. The molecules includes phthalimid-like structures and bear the potential to release chlorinated phthalimid after enzymatic or bacterial cleavage. Therefore, the analogue substance was choosen to examine toxicity after repeated dose administration and toxicity to reproduction and development.

Procedure and observations

The test item (analogue) 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 (drinking water served as vehicle), 100 mg/kg bw/d, 300 mg/kg bw/d and 1000 mg/kg bw/d. 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. A detailed clinical observation was performed in all animals. Body weights and food consumption were determined in F0 animals. Clinicochemical and hematological examinations as well as urinalyses and FOB were performed in all animals towards the end of the administration period. All animals were assessed by gross pathology; weights of selected organs were recorded and a histopathological examination was performed. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed with CO2 and examined macroscopically for external and visceral findings.

Clinical examinations, reproductive performance, clinical pathology, histopathology and gross pathology did not reveal any finding in treated animals. Clinical examination and gross pathology did not reveal any effect on pups.

Discussion

Thus, 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.

Justification for classification or non-classification

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for reproductive toxicity under Regulation (EC) No. 1272/2008, as amended for the second time in Directive (EC 286/2011).