Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
Repeated Dose and Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 15-10-2010 to 31-03-2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
- Purity: 99.3%

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Co., Ltd
- Age at study initiation: 9 weeks
- Weight at study initiation: 476.7± 33.3g (males) and 286.0± 20.2 (female) at Day 0 of test item adminstration
- Fasting period before study: 16 hrs
- Housing: Individual in aluminum front and floor stainless steel mesh breeding cage
- Diet: Radiation sterilized solid feed (CRF-1, Lot No. 100203, 100302, Oriental Yeast Co.) ad libitum
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.8 to 23.2 ° C
- Humidity (%): 53.8 to 64.8%
- Air changes (per hr): 12 times per hr
- Photoperiod (hrs dark / hrs light): 12 hrs light/dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% methyl cellulose aqueous solution (suspended)
Details on oral exposure:
VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Males: 42 days
Females; 42-52 days (from 14 days before mating to day 4 of lactation)
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
240 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Males: 7 rats/group (control and high dose groups of main study)
+5 rats/group (control and high dose groups of recovery).
12 rats/group (low and middle dose groups of main study).

Females: 12 rats/group (all groups of main study)
+ 5 rats/group (control and high dose groups of recovery).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In the 14-day range finding study with 6 males and 6 females at doses of 0, 20, 60, 200, 600 mg/kg bw/day, following findings were reported.

600 mg/kg bw/day: Death (M6/6, F6/6), diarrhea, mucous stool, soiled fur, ptosis (MF), body weight↓(MF), food consumption↓(MF), enlarged lumen of the stomach, red or black
patches in the stomach, enlarged lumen of the small intestine, liquid content in the stomach and the large intestine, reddish content in the small and large
intestine, atrophy of the thymus, blackish discoloration of the large intestine, brownish discoloration of the lymph node, red patch / edema of the thymus,
enlarged adrenal, emaciation (MF)

200 mg/kg bw/day: Diarrhea, suppressed body weight gain, enlarged lumen of the stomach / small intestine / large intestine, atrophy of the spleen and the thymus (F1/6), Hct↓,
Hgb↓, RBC↓(M), TP↓, Alb↓, BUN↓ (M), monocyte↑(F), red or brown patches in the mesenteric lymph node (MF)

60 mg/kg bw/day: Hct↓, Hgb↓, RBC↓ (M), TP↓, Alb↓, BUN↓(M)

20 mg/kg bw/day: No effect

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: Yes

URINALYSIS: Yes (males only)

Behaviour: Yes (Functional Observational Battery:detailed observation, sensory reactivities, grasping power, motor activity).

Sacrifice and pathology:
GROSS PATHOLOGY: Yes and organ weights

HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Death occurred in 4 females and 2 males at 240 mg/kg bw/day. These animals showed loose stools, diarrhea, abnormal respiration noise, irregular respiration, etc. before death.

In animlas that survived, salivation was observed in males in the 60 mg/kg group and in animals of both sexes in the 240 mg/kg group.
Mortality:
mortality observed, treatment-related
Description (incidence):
Death occurred in 2 males and 2 females in the 240 mg/kg group of the main study group and 2 females in the 240 mg/kg group of the satellite group.
Body weight and weight changes:
no effects observed
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:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Inorganic phosphorus and blood potassium concentration were decreased in females in the 240 mg/kg group of the satellite group; these changes were considered to be related to the dysfunction of the digestive tract by the test substance.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Total excretion volume of potassium and urinary potassium concentration were decreased, urinary pH was lowered and urinary protein was slightly increased in the 240 mg/kg group, total protein, albumin and α1 globulin were decreased and an α2 globulin ratio was increased in males in the 240 mg/kg group of the main study group, inorganic phosphorus was decreased in females in the 60 and 240 mg/kg groups of the main study group; these changes were considered to be related to the dysfunction of the digestive tract by the test substance.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Death occured in 4 females and 2 males at 240 mg/kg bw/day. At necropsy, dilatation of the gastric lumen with gas and/or liquid contents was observed in all of the above animals, and brownish mesenteric lymph nodes, red patches in the stomach, dilatation of the lumen of the small intestine with gas and/or liquid red contents and dilatation of the lumen of the large intestine (cecum) with gas contents in some of them.

In the pathological examination of animals that survived, brownish mesenteric lymph nodes, black patches in the glandular stomach, dilatation of the intestinal lumen (duodenum, jejunum and cecum) with gas contents and gas contents in the large intestine (colon) were observed, and these were considered to reflect gastric injury and dysfunction of the digestive tract by the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathologically, blood absorption by the mesenteric lymph nodes was observed in all of the animals which were found dead, and brown pigments in the mesenteric lymph nodes, hemorrhage from the forestomach, edema, polymorphonuclear cell infiltration and inflammation in the glandular stomach in some of them. From these pathological findings, the cause of death was considered to be gastric injury and dysfunction of the digestive tract by the test substance.


The following histopathological findings also confirmed the presence of the gastric injury by the test substance in animals that survived: blood absorption by the mesenteric lymph nodes, brown pigments in the mesenteric lymph nodes and hemorrhage from the forestomach in males or females in the 60 or 240 mg/kg group.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathologically, squamous cell hyperplasia in the forestomach was noted in 4/4 dead females at 240 mg/kg bw/day.

Squamous cell hyperplasia and an increase in apoptosis in the forestomach, proliferation of surface mucous cell in the glandular stomach in males or females in the 60 or 240 mg/kg group. An increase in mitosis in the glandular stomach was observed in animals of both sexes given 15 mg/kg or more. The latter finding was considered to be an effect of the test substance but was concluded to be toxicologically meaningless because there were no other findings suggestive of gastric injury in those groups.

Other effects:
not examined
Details on results:
Please refer to Figures 1-6, Tables 1- 16 and Appendices 1 - 18 for all relevant data.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
< 15 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
histopathology: neoplastic
Dose descriptor:
NOAEL
Effect level:
ca. 15 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
histopathology: neoplastic

Target system / organ toxicity

open allclose all
Critical effects observed:
yes
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
60 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
mesenteric lymph node
Treatment related:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
240 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
intestine
mesenteric lymph node
Treatment related:
yes

Applicant's summary and conclusion

Conclusions:
In a repeated dose and reproduction/developmental toxicity screening test with 1,1'-(1,3-phenylene)bis-1H-pyrrole-2,5-dione in Crl:CD (SD) rats, the NOAEL for both sexes was 15 mg/kg bw/day and the NOEL was <15 mg/kg bw/day.
Executive summary:

In a repeated dose and reproduction/developmental toxicity screening test (C545 (314 -026)), 1,1'-(1,3-phenylene)bis-1H-pyrrole-2,5-dione (99.3%) was administered to four groups of Crl:CD (SD) rats (12 animals/sex/group) by gavage in 0.5% methyl cellulose at dose levels of 0, 15, 60 and 240 mg/kg bw/day 7 days per week for 42 days (males) and 42-52 days (females).

Death occurred in 4 females and 2 males at 240 mg/kg bw/day. These animals showed loose stools, diarrhea, abnormal respiration noise, irregular respiration, etc. before death. In animals that survived, salivation was observed in males in the 60 mg/kg group and in animals of both sexes in the 240 mg/kg group. Total excretion volume of potassium and urinary potassium concentration were decreased, urinary pH was lowered and urinary protein was slightly increased in the 240 mg/kg group, total protein, albumin and α1 globulin were decreased and an α2 globulin ratio was increased in males in the 240 mg/kg group of the main study group and inorganic phosphorus was decreased in females in the 60 and 240 mg/kg groups of the main study group. Inorganic phosphorus and blood potassium concentration were decreased in females in the 240 mg/kg group of the satellite group; these changes were considered to be related to the dysfunction of the digestive tract by the test substance. There were no effects on body weight, food consumption, hematologic parameters, blood coagulation or organ weight.

At necropsy, dilatation of the gastric lumen with gas and/or liquid contents was observed in all animals that died, and brownish mesenteric lymph nodes, red patches in the stomach, dilatation of the lumen of the small intestine with gas and/or liquid red contents and dilatation of the lumen of the large intestine (cecum) with gas contents in some of them. In the pathological examination of animals that survived, brownish mesenteric lymph nodes, black patches in the glandular stomach, dilatation of the intestinal lumen (duodenum, jejunum and cecum) with gas contents and gas contents in the large intestine (colon) were observed, and these were considered to reflect gastric injury and dysfunction of the digestive tract by the test substance.

Histopathologically, blood absorption by the mesenteric lymph nodes was observed in all of the animals which were found dead, and brown pigments in the mesenteric lymph nodes, hemorrhage from the forestomach, edema, polymorphonuclear cell infiltration and inflammation in the glandular stomach in some of them. From these pathological findings, the cause of death was considered to be gastric injury and dysfunction of the digestive tract by the test substance. Squamous cell hyperplasia in the forestomach was noted in 4/4 dead females at 240 mg/kg bw/day. The following histopathological findings also confirmed the presence of the gastric injury by the test substance in animals that survived: blood absorption by the mesenteric lymph nodes, brown pigments in the mesenteric lymph nodes, squamous cell hyperplasia and an increase in apoptosis in the forestomach, hemorrhage from the forestomach, proliferation of surface mucous cell in the glandular stomach in males or females in the 60 or 240 mg/kg group. An increase in mitosis in the glandular stomach was observed in animals of both sexes given 15 mg/kg or more. This finding was considered to be an effect of the test substance but was concluded to be toxicologically meaningless because there were no other findings suggestive of gastric injury in those groups.

The NOAEL for both sexes was 15 mg/kg bw/day and the NOEL was <15 mg/kg bw/day.

This repeated dose and reproduction/developmental toxicity screening study in the rat is acceptable and satisfies the guideline requirement for this oral study (OECD 422) in rats.