Pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis[4-(1,1-dimethylethyl)phenyl]-2,5-dihydro-

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD testing guideline study with well characterized material

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: (P) Approximately 11 weeks.
- Weight at study initiation: (P) Males: 297 - 321 g; Females: 195 - 215 g
- Housing: groups of 5 animals/sex/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24.4°C
- Humidity (%):40 - 70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 May 2012 (allocation) To:4 July 2012

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was
made for specific gravity/density of the test substance, vehicle, and/or formulation.

VEHICLE

- Concentration in vehicle: adjusted for dose and volume
- Amount of vehicle (if gavage): 5 mL/kg body weight

Details on mating procedure:

- M/F ratio per cage: one-to-one
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal
copulatory plug. referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): single cages

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dose preparations were taken on a single occasion during the treatment phase.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 42-50 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).

Frequency of treatment:

Once daily for 7 days per week

Details on study schedule:

not appliecable for screening study

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on results of a 28-day study.

Positive control:

not required.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.


Food consumption: Weekly, except for males and females which were housed together for mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

Oestrous cyclicity (parental animals):

not investigated

Sperm parameters (parental animals):

spermatogenic staging profile for high dose and control animals

Litter observations:

STANDARDISATION OF LITTERS
not relevant for screening study

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, presence of milk in stomach

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined as far as possible for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, following completion of the mating period (29 days of dose administration).
- Maternal animals: All surviving animals, lactation days 5 -7

GROSS NECROPSY
- Gross necropsy consisted of macroscopic examination of the cranial, thoracic and abdominal tissues
and organs, with special attention being paid to the reproductive organs

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination:


Cervix
Preputial gland
Clitoral gland
Prostate gland
Coagulation gland
Seminal vesicles
Epididymides
Testes
Mammary gland area
Uterus
Ovaries
Vagina
All gross lesions


The following slides were examined by a pathologist:
- The ovaries, testes and epididymides of the animals of Groups 1 and 4.
- The additional slides of the testes of the males of Groups 1 and 4, and all males suspected to be infertile, to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs* of animal nos. 7 and 47 (Group 1, female was not pregnant) and 11 and 51 (Group 2, female had a total litter loss)
* Reproductive organs includes the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate
gland, seminal vesicles, testes, uterus, and vagina.

Organ weights were determined for epididymides and testes.

The numbers of former implantation sites and corpora lutea were recorded for all paired females.

Postmortem examinations (offspring):

Clinical signs,
Body weight
Macroscopical investigation

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to- one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

Reproductive indices:

Duration of gestation
Gestation index (%)
Conception index (%)
Fertility index (%)
Mating (%)

Offspring viability indices:

Percentage live males at First Litter Check:
Number of live male pups at First Litter Check x 100 / Number of live pups at First Litter Check

Percentage live females at First Litter Check:
Number of live female pups at First Litter Check x 100 / Number of live pups at First Litter Check

Percentage of postnatal loss at Days 0-4 of lactation:
Number of dead pups on Day 4 of lactation x 100 /Number of live pups at First Litter Check:

Viability index:
Number of live pups on Day 4 post-partum x 100 / Number of pups born alive

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Organ weights and haematology parameters did not differ from those of control animals.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Reproductive effects observed:

not specified

Table 1: REPRODUCTION DATA SUMMARY

	control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Females paired	10	10	10	10
Females mated	10	10	10	10
Females pregnant	10	9	9	10
Non-pregnant females	0	1	1	0
Females with implantation sites only	0	1	0	0
Females found dead	1	0	0	0
Females with living pups on Day 1	9	8	9	10
Mating index (%) (Females mated / Females paired) * 100	100.0	100.0	100.0	100.0
Fertility index (%) (Pregnant females / Females paired) * 100	100.0	90.0	90.0	100.0
Conception index (%) (Pregnant females / Females mated) * 100	100.0	90.0	90.0	100.0
Gestation index (%) (Females with living pups on Day 1 / Pregnant females) * 100	90.0	88.9	100.0	100.0

+/++ Steel-test significant at 5% (+) or 1% (++) level

Table 2: CORPORA LUTEA AND IMPLANTATION SITES SUMMARY

		control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Corpora Lutea	MEAN	13.2	11.1	12.7	12.0
	ST.DEV	2.4	4.1	1.7	1.6
	N	10	9	9	10
Implantations	MEAN	11.7	10.3	10.8	11.3
	ST.DEV	1.7	3.6	2.0	1.5
	N	10	9	9	10

+/++ Steel-test significant at 5% (+) or 1% (++) level

Table 3 DEVELOPMENTAL DATA (DEVELOPMENTAL DATA)

		control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
LITTERS
TOTAL		9	8	9	10
DURATION OF GESTATION
MEAN (+)		21.2	21.5	21.1	21.2
ST.DEV.		0.4	0.5	0.3	0.4
N		9	8	9	10
DEAD PUPS AT FIRST LITTER CHECK
LITTERS AFFECTED (#)		1	0	2	0
TOTAL		1	0	2	0
MEAN (+)		0.1	0.0	0.2	0.0
ST.DEV.		0.3	0.0	0.4	0.0
N		9	8	9	10
LIVING PUPS AT FIRST LITTER CHECK
% OF MALES / FEMALES (#)		51 / 49	42 / 58 #	53 / 47	54 / 46
TOTAL		102	89	88	109
MEAN (+)		11.3	11.1	9.8	10.9
ST.DEV.		1.4	2.1	1.9	1.4
N		9	8	9	10
POSTNATAL LOSS % OF LIVING PUPS		1.0	1.1	1.1	0.9
LITTERS AFFECTED (#)		1	1	1	1
TOTAL (#)		1	1	1	1
MEAN (+)		0.1	0.1	0.1	0.1
ST.DEV.		0.3	0.4	0.3	0.3
N		9	8	9	10
VIABILITY INDEX (#)		99.0	98.9	98.9	99.1

Viability index = (Number of alive pups before planned necropsy / Number of pups born alive) *100

+/++ Steel-test significant at 5% (+) or 1% (++) level

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

Table 4 PRECOITAL TIME

Days of the pairing period	control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
1	1	3	1	1
2	5	3	4	3
3	3	2	4	4
4	1	2	1	2
MEDIAN PRECOITAL TIME	2	2	3	3
MEAN PRECOITAL TIME	2	2	3	3
N	10	10	10	10

Table 5: BODY WEIGHTS OF PUPS (GRAM)

DAY	SEX		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
1	M	MEAN	6.1	6.5	6.3	6.3
		ST.DEV.	0.3	0.9	0.8	0.4
		N	9	8	9	10
	F	MEAN	5.9	6.1	6.0	6.0
		ST.DEV.	0.4	0.9	0.7	0.5
		N	9	8	9	10
	M+F	MEAN	6.0	6.2	6.1	6.2
		ST.DEV.	0.3	0.9	0.7	0.4
		N	9	8	9	10
4	M	MEAN	8.9	9.7	9.2	9.3
		ST.DEV.	0.5	1.7	1.6	0.8
		N	9	7	9	10
	F	MEAN	8.7	9.2	8.8	8.8
		ST.DEV.	0.7	1.7	1.4	0.8
		N	9	7	9	10
	M+F	MEAN	8.9	9.4	9.0	9.0
		ST.DEV.	0.6	1.7	1.5	0.8
		N	9	7	9	10

Conclusions:

No adverse effects were observed up to the limit dose of 1000 mg/kg bw.

Executive summary:

In the OECD 421 screening study for reproductive and developmental toxicity, treatment by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg body weight/day revealed no parental, reproductive or developmental toxicity up 1000 mg/kg body weight/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

valid without restrictions

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Based on available toxicity data of the main component (NOAEL = 1000 mg/kg bw/day, 28 days). The dose levels for this reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg bw/day. The study was based on the guideline OECD 421, Reproduction/Developmental Toxicity Screening Test, July 1995 and GLP. After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 100, 300 and 1000 mg/kg bw/day. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 40-45 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), body weight and food consumption (at least at weekly intervals), haematology and clotting tests (Week 4, males only), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Formulations were analyzed once during the study to assess accuracy, homogeneity and stability.

Accuracy, homogeneity and stability of formulations were demonstrated by analyses. No parental, reproduction and developmental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day).

Short description of key information:
The substance did not cause toxicity to reproduction in rat at the screening information level at the limit dose (OECD 421, GLP) (BASF 2012).

Justification for selection of Effect on fertility via oral route:
only study available.

Effects on developmental toxicity

Description of key information

No indication of developmental toxicity in rats was observed in a screening study (OECD 421, GLP) at the limit dose of 1000 mg/kg bw (BASF 2012).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

valid, but screening study.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Based on available toxicity data of the main component (NOAEL = 1000 mg/kg bw/day, 28 days). The dose levels for this reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg bw/day. The study was based on the guideline OECD 421, Reproduction/Developmental Toxicity Screening Test, July 1995 and GLP. After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 100, 300 and 1000 mg/kg bw/day. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 40-45 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), body weight and food consumption (at least at weekly intervals), haematology and clotting tests (Week 4, males only), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Formulations were analyzed once during the study to assess accuracy, homogeneity and stability.

Accuracy, homogeneity and stability of formulations were demonstrated by analyses. No parental, reproduction and developmental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day).

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available screening study is considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for fertility or developmental toxicity under Directive 67/548/EEC, as amended for the 31st time in Directive2009/2/EG. 

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

The available screening study is reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for fertility or developmental toxicity under Regulation (EC) No. 1272/2008, as amended for the third time in Directive (EC 618/2012).

During the four days covered in the screening study, no effects via lactation were observed.