Pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis([1,1'-biphenyl]-4-yl)-2,5-dihydro-

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6.4.2011 - 3.11.2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted July 27th 1995

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid

Details on test material:

Red powder
storage at room temperature
unlimited stability

Specific details on test material used for the study:

Red powder
storage at room temperature
unlimited stability

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding, VELAZ, Czech Republicc
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 10 weeks
- Weight at study initiation: mean 319 g (males), 210g (females)
- Fasting period before study: no
- Housing: 2 of the same sex (premating), 1 male and 1 female (mating), single caging (pregnancy), dams with litter

- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

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

IN-LIFE DATES: From: 27.2.2011 (animal arrival) To: 31.5.2011 (males) and 26.6.2011 (females)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Daily preparation of test substance dosing solution

VEHICLE
- Justification for use and choice of vehicle (if other than water): Water with 0.5% methylcellulose to generate a stable dispersion
- Concentration in vehicle: adjusted to 1ml per 100 g body weight

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy
- Further mating after unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individual cages
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The substance is an insoluble pigment for which a substance-specific analytical method was not available.

Duration of treatment / exposure:

4 weeks (males)
6 weeks (females)

Frequency of treatment:

Daily

Details on study schedule:

males and females - 2 weeks prior to the mating period and during the mating period,
pregnant females - during pregnancy and till the 3rd day of lactation,
males - after mating period - totally for 28 days,
non-pregnant females (mated females without parturition) - for 25 days after the confirmed mating.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

160 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

400 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: 1000 mg/kg bw is the limit dose in the OECD testing guideline. A 14-day dose-range finding study was performed with 125, 250, 500 and 1000 mg/kg bw with each 5 males and females per dose group.
- Fasting period before blood sampling for clinical biochemistry: not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: males weekly, females weekly in premating and mating, during pregnancy day 0,7,14,20th day and during lactation on day 0, 1 and 4

FOOD CONSUMPTION:
- same schedule as body weight


OTHER:
Vaginal smears daily in mating period

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Sperm samples were taken from one epididymis and sperm morphology was assessed. All deviations - (eg broken tail, abnormal form of tail, double head, amorphous head, no head, abnormal form of neck) - were recorded.

Litter observations:

Number and sex of pups, stillbirths, live births and presence of gross anomalies
daily observation of behavioural abnormalitites until postnatal day 4

Postmortem examinations (parental animals):

Absolute and relative weights of testes, epididymis, prostate gland and pituitary gland, ovaries, uterus

Histopathology of relevant gross lesions, pituitary gland, coagulation gland, prostate gland, seminal vesicles, epididymis, testes, cervix of uterus, ovaries, uterau, vagina

Postmortem examinations (offspring):

Gross anomalies
Sex
content of milk in stomach

Statistics:

ANOVA test (QC.Expert 2.5)

Reproductive indices:

Male mating index
Female mating index

Male fertility index
Female fertility index

Gestation index

Offspring viability indices:

Pup survival index (number of live pups on day 4 post partum x 100/number of pups born alive (incl dead pups with aerial lungs)

Pre-implantation loss (Number of corpus lutea - number of implantations)
Post-implantation loss (Number of implantations - number of live births)
Post-natal loss (Number of live births - number of alive at postnatal day 4)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

excrements coloured by the test substance (two rats in the low dose group, seven rats in the mid dose and 13 rats of the high dose group)

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

See tables 1 and 2

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

see tables 3 and 4

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effect up to the limit dose.

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effect up to the limit dose.

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1: Body weight of males mean) in g
Dose level (mg/kg bw)	0	160	400	1000
week 0	319.65	320	(321.13	319.73
week 1	359.99	359.88	363.69	359.73
week 2	384.48	382.49	389.4	386.93
week 3	402.86	396.01	406.89	402.37
week 4	421.09	411.02	423.68	418.33

Table 2

Body weight of females (mean) in g
	Dose level (mg/kg bw)	0	160	400	1000
premaing	week 0	248.59	240.48	243.08	243.33
	week 1	228.81	229.18	221.84	223.18
	week 2	238.42	235.21	231.66	231.85
gestation	day 0	248.59	240.48	243.08	243.33
	day 7	265.84	261.75	268.25	265.03
	day 14	290.81	287.74	295.42	296.04
	day 20	347.69	359.06	351.17	354.29
lactation	day 0/1	281.91	279.45	284.37	282.13
	day 4	309.83	298.54	303.30	305.93

Table 3

Microscopical findings in males
Dose level (mg/kg bw)	0	160	400	1000
number of animals	12	12	12	12
Pituitary gland: small cysts	0	0	1	0
Testes: less than 10% tubules; degeneration and/or atrophy of germinative epithelium	2	7	4	3
Epididymides: interstitium and epithelium - slight lymphocyte infiltration	12	12	12	12
Prostate gland - atrophy of alveolar epithelium	0	0	1	0
Prostate gland - lymphocyte infiltration - sporadic	3	1	2	2
Prostate gland: functional hyperplasia	0	2	0	0

Table 4

Microscopic findings in females
Dose level (mg/kg bw)	0	160	400	1000
number of animals	12	12	12	12
animals without findings	6	4	4	3
Pituitary gland: cysts	0	0	0	1
Ovaries: follicular and/or luteal cysts	0	2	3	1
Uterus: signs of previous gravidity - foci of siderphages and/or liphages and/or haemorrhage	5	5	4	4
Uterus: hydrometra	0	0	3	1
Uterus: proliferation of endometrial stroma	0	0	1	0
Vagina: cell detritus in lumen	3	3	2	2
Vagina: mononuclear infiltration of mucosa	0	0	1	0

Applicant's summary and conclusion

Conclusions:

The test substance is not toxic to reproduction in rats at the screening level (OECD 421, GLP).

Executive summary:

The test substance was tested for its potential to cause toxicity to reproduction in rats in a GLP-conform screening test according to OECD guideline 421. The study was performed with gavage doses of 140, 400 and 1000 mg/kg bw using carboxymethyl cellulose as vehicle. Rats were treated prior to mating and during mating. After mating, dams were kept during gestation and until postnatal day 4. The growth (body weight and food consumption) and clinical status of parental animals were not adversely influenced by the test substance treatment, so that no toxic effect of the test substance to parental animals was observed. Biometry and structure of reproductive organs of parental males and females and quality of sperms of parental males were not adversely influenced by the test substance treatment. The ability of male and female animals to successfully mate and produce viable offspring was unaffected by the test substance treatment. The development of pups was not changed in treatment groups. Test-substance coloured chymus in stomatch and/or intestine was registered in rats of all dose levels. Feces also showed the colour of the test substance. No adverse effects on systemic toxicity, reproductive toxicity or pub development were observed up to the highest tested dose. Thus, the NOEL was 1000 mg/kg bw.