4,4'-diamino[1,1'-bianthracene]-9,9',10,10'-tetraone

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:

2011 - 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline and GLP compliant 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:

other: Cri :WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 11 - 12 weeks
- Fasting period before study: overnight
- Housing: 1 rat per cage, except during mating
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2012-02-14 To: 2012-04-19

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
To prepare the suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a magnetic stirrer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least twice a week and were stored at room temperature.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning (Mating attempts were performed for a maximum of 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 their litters were housed together until PND 4 (end of lactation). Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.

The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in drinking water was demonstrated over a period of 7 days at room temperature. The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations (deviations <5%).

Duration of treatment / exposure:

The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice (males: 34 / 35 days; females: 49 days)

Frequency of treatment:

daily

Details on study schedule:

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

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: Range-finder study

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied. A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before initial test substance administration and thereafter at weekly intervals.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of F0 parents were determined on study day 0 (start of the administration period) and thereafter once a week, in males throughout the study and in females during premating and mating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, after the day of parturition (postnatal day [PND] 0) and on PND 4.


FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

Food consumption was determined once a week for male and female parental animals, with the following exceptions:

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.

Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OTHER:
The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.

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, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: all surviving animals
- Maternal animals: all surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS:
Weight assessment was carried out on all animals. The following weights were determined:
Adrenal glands, Anesthetized animals, Brain, Epididymides, Heart, Kidneys, Liver, Testes, Spleen, Thymus

The following organs / tissues were preserved in neutral-buffered 4% formaldehyde or in modified Davidson’s solution:
Adrenal glands, All gross lesions, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Eyes with optic nerve (modified Davidson’s solution), Esophagus, Extraorbital lacrimal gland, Epididymides (modified Davidson’s solution), Female mammary gland, Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Target organs, Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina

Postmortem examinations (offspring):

SACRIFICE
- 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.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Statistics:

Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter: Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means; Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions; Proportions of affected pups per litter with necropsy observations: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.

Reproductive indices:

Male / female mating indices, male / female fertility indices, gestation index, live birth index, postimplantation loss

Offspring viability indices:

Viability index was calculated using the number of live pups/litter on the day after birth, and on lactation day 4.

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: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

During premating, mating and post-mating red discolored feces was seen in all animals of the test groups 1-3 (100, 300 and 1000 mg/kg bw/d). The sign came up first on study day 7 in test group 1 (100 mg/kg bw/d) and on study day 1 in test groups 2 and 3 (300 and 1000 mg/kg bw/d). The findings were considered to be related to treatment as the substance is a red pigment, but assessed as being non-adverse.

For all F0 parental males, which were placed with females to generate F1 pups, mating was confirmed with the exception of male control animal No. 1 and high-dose male No. 31. Thus, the male mating index was 90% in control group and test group 3 (0 and 1000 mg/kg bw/d) and 100% in test groups 1 and 2 (100 and 300 mg/kg bw/d). Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. Three males of control group (animal Nos. 1, 4 and 8 mated with female Nos. 101, 104 and 108, respectively) and 1 male animal of test group 3 (animal No. 31 mated with female No. 131) did not generate F1 pups. Thus, the male fertility index ranged between 70% and 100%. These values 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 .

The female mating index calculated after the mating period for F1 litter was 90% in control group and test group 3 (0 and 1000 mg/kg bw/d) and 100% in test groups 1 and 2 (100 and 300 mg/kg bw/d). The mean duration until sperm was detected (GD 0) was 2.8, 3.3, 2.3 and 2.6 days in test groups 0-3 (0, 100, 300, 1000 mg/kg bw/d, respectively).

The mean duration of gestation was between 22.0 and 22.4 days and did not show significant differences.

The gestation index reached 100% in all groups including the control.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of effects up to the limit dose

Critical effects observed:

no

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

The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 100% (control group), 99% (test groups 1 and 2) and 93% (test group 3). Single stillborn pups were seen in test groups 1 and 2 and in one female of test group 3. Additionally, 2 more dams of test group 3 had 2 and 5 stillborn pups.

The viability index as indicator for pup mortality between PND 0 and 4 was 99% for the control, 98.2% for test group 1 (100 mg/kg bw/d), 99.1% for test group 2 (300 mg/kg bw/d) and 93.5 % for test group 3 (1000 mg/kg bw/d).

The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences between test groups.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects up to the limit dose.

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

The substance did not cause adverse effects on reproductive function in the screening study (OECD 422) in rats at the limit dose.

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

Quality of whole database:

Valid

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Pigment Red 177 was administered orally by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 100, 300 and 1000 mg/kg bw/d. The study followed OECD testing guideline 422 and GLP (BASF 2013). The test substance was suspended in water as it is not soluble in any vehicle.

Regarding clinical examinations,signs of general systemic toxicity were not observed in male and female parental animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) during the entire study period. Female animal No. 138 of test group 3 (1000 mg/kg bw/d) showed clinical findings during lactation and did not properly nurse its pups on postnatal day (PND) 0 and 1. Taking into account that the litter size of 17 pups was very high for this rat strain, giving birth to this high amount of pups was considered to have been exhausting and stressful for the animal. The resulting impaired clinical condition of the animal during lactation was assessed to have been influenced the nursery and, given that, led to the increased pup mortality. In addition, the increased occurrence of runts on PND 1 was assumed as being related to the litter size which has been proven to have an impact on the development status of pups (Romero et al., 1992). Therefore, the occurrence of the mentioned findings in the single animal was not assessed as being related to treatment. Fertility indices for male and female animals were not impaired by test-substance administration even at a dose level of 1000 mg/kg bw/d. In addition, live birth indices of pups in all test groups were not influenced.

Concerning clinical pathology no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. Regarding pathology, there were no treatment-related organ weight changes. Histopathology revealed the presence of the test substance in the lumen of the gastrointestinal tract in form of red particles or crystals. This correlated with the red discoloration of the contents observed macroscopically. This finding did not cause any tissue alterations and therefore it was considered to be treatment-related but not adverse.

As discussed in the toxicokinetics section, the pigment is considered to be not taken up by the body by the oral route.

Short description of key information:
No adverse effects on fertility were observed in a screening study in rats (OECD 422, BASF 2013). The highest tested dose was 1000 mg/kg bw.

Justification for selection of Effect on fertility via oral route:
key study

Effects on developmental toxicity

Description of key information

No adverse findings on offspring were observed in a screening study for developmental toxicity in rats with doses of up to 100, 300 and 1000 mg/kg bw (OECD 422, GLP) (BASF 2013).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

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 compliant

Qualifier:

according to guideline

Guideline:

other: OECD 422

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Cri :WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 11 - 12 weeks
- Fasting period before study: overnight
- Housing: 1 rat per cage, except during mating
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2012-02-14 To: 2012-04-19

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
To prepare the suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a magnetic stirrer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least twice a week and were stored at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

PREPARATION OF DOSING SOLUTIONS:
To prepare the suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a magnetic stirrer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least twice a week and were stored at room temperature.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning (Mating attempts were performed for a maximum of 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 their litters were housed together until PND 4 (end of lactation). Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.

The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.

Duration of treatment / exposure:

The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice (males: 34 / 35 days; females: 49 days)

Frequency of treatment:

daily

Duration of test:

The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice (males: 34 / 35 days; females: 49 days)

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: Range-finder study

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied. A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before initial test substance administration and thereafter at weekly intervals.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of F0 parents were determined on study day 0 (start of the administration period) and thereafter once a week, in males throughout the study and in females during premating and mating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, after the day of parturition (postnatal day [PND] 0) and on PND 4.


FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

Food consumption was determined once a week for male and female parental animals, with the following exceptions:

Food consumption was not determined during the mating period
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.

Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

Ovaries and uterine content:

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

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

Statistics:

Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions. Siegel S. (1956): Non-parametric statistics for behavioural sciences. McGraw-Hill, New York

Indices:

Live birth index, Postimplantation loss

Historical control data:

presented in the report

Details on maternal toxic effects:

Maternal toxic effects:no effects

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

No adverse effects on offspring were observed in a screening study in rats (OECD 422) at the limit dose of 1000 mg/kg bw.

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

The study is discussed in the section for fertility and repeated dose toxicity.

The viability index as indicator for pup mortality was not impaired by test-substance administration at a dose level of 1000 mg/kg bw/d. The postimplantation loss was comparable for all dose groups. No adverse effects on offspring were observed.

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.