Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 Aug 2021 - 07 Nov 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted July 2016
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,9-dimethylanthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone
EC Number:
226-866-1
EC Name:
2,9-dimethylanthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone
Cas Number:
5521-31-3
Molecular formula:
C26H14N2O4
IUPAC Name:
2,9-dimethylisoquino[4',5',6':6,5,10]anthra[2,1,9-def]isoquinoline-1,3,8,10(2H,9H)-tetrone
Test material form:
solid: nanoform, no surface treatment
Details on test material:
- State of aggregation: solid, powder
- Particle size distribution (TEM): 64.9 nm (D50)
- Mass median aerodynamic diameter (MMAD): not specified
- Geometric standard deviation (GSD): not specified
- Shape of particles: cubic, spherical, rod
- Surface area of particles: 29.5 m²/g
- Crystal structure: crystalline
- Coating: no
- Surface properties: not applicable
- Density: 1600 kg/m³ at 20°C
- Moisture content: refer to IUCLID chapter 1
- Residual solvent: refer to IUCLID chapter 1
- Activation: not applicable
- Stabilisation: not applicable

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
(CD:SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, NC)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 12-13 wks
- Weight at study initiation: (P) Males: 355.1 - 502.1 g; Females: 225.8 to 278.6 g
- Housing: group housed, males were individually housed three days prior to cohabitation
- Diet: Purina Certified Rodent Diet, LabDiet 5002, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature: 71 to 73 °F (ca. 21.7 - 22.8 °C)
- Humidity: 45 to 63 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: August 10, 2021 To: November 7, 2021

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was prepared at concentrations of 20, 60, and 200 mg/mL. The prepared test article was stored refrigerated (2-8 °C) in accordance with the results of the pre-study stability assessment and residual formulations were disposed of after the expiration date.

VEHICLE
- Concentration in vehicle: 20, 60, and 200 mg/mL
- Amount of vehicle (if gavage): 6.5 mL/kg (Group 1,3,4) or 7.5 mL/kg (Group 2)
- Purity: Deionized water
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: max. 14 d
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
- Start of mating: Study day 15
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for analysis of stability were collected from formulations prepared on August 25,
2021, stored at room temperature, transferred to the designated test site (at room temperature)
and analyzed after 7 and 28 days. Aliquots from each storage sample were analyzed for
concentration and the results compared to the concentration determined initially. Dose
formulations were transferred to the designated test site (at room temperature) and analyzed
for test article concentration using a non-GLP validated procedure.
In addition, the formulations of first preparation were analyzed to confirm
homogeneity. The final preparation was analyzed for test article concentration; however,
homogeneity was not evaluated from the final preparation as an aliquot was not collected for
this task.
Formulations for use, analysis, and residuals were stored refrigerated (2 to 8°C) in accordance
with the results of the pre-study stability assessment.
Duration of treatment / exposure:
- Males: Beginning from day 1 until the day prior to termination (30 d)
- undelivered females: until GD 23
- delivered females: until PND 13
- Pups: not directly dosed
Frequency of treatment:
daily, 7 days a week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control (Group 1)
Dose / conc.:
100 mg/kg bw/day
Remarks:
Low (Group 2)
Dose / conc.:
300 mg/kg bw/day
Remarks:
Mid (Group 3)
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
High (Group 4)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Prior to blood collection for clinical pathology evaluation, F0 animals were fasted overnight (approximately 12 to 22 hours).

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (moribundity and mortality); daily (Cage side observation)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly and for dams on GD 0, 7, 14, 21 and PND 7 and 14
- Dams were evaluated for reduced maternal care or abnormal maternal behavior beginning on PND 0.

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly, beginning on day 1, and on the day of termination (fasted body weight)
- Dams were weighed on GD 0, 7, 14, and 21, and on PND 1, 4, 7, and 13

FOOD CONSUMPTION:
- Quantitative food consumption parameters were recorded for F0 animals during the premating and post-mating periods (including gestation and lactation periods) to coincide with body weight collection intervals. (given in g/Day)

NEUROBEHAVIORAL ASSESSMENTS:
- No. of animals: 5 animals per sex per dose
- Animal selection: The first 5 males per group were utilized, and 5 females per group were selected based on their parturition date.
- Time schedule: Day 29 (males) and PND 11 +/- 1 (females)
- Parameters tested:
> Spontaneous Locomotor Activity
> Functional Observational Battery
> Grip Strength

CLINICAL PATHOLOGY:
- No. of animals: 5 animals per sex per dose
- Time schedule: Males: on Day of termination (Day 31); Females: PND 14
- Anesthetized with carbon dioxide/oxygen (CO2/O2, 70/30%)
- Parameters examined:
> Haematology (Erythrocyte count, Hemoglobin, Hematocrit, Leukocyte count (total), Leukocyte differential (absolute), Platelet count, Reticulocyte count (absolute), Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration)
> Coagulation (Activated partial thromboplastin time, Fibrinogen, Prothrombin time)
> Serum chemistry (Alanine aminotransferase, Albumin, Aspartate aminotransferase, Bile acids, Bilirubin, Blood urea nitrogen, Calcium, Chloride, Cholesterol, Creatinine, Glucose, Inorganic phosphate, Potassium, Sodium, Total protein)

THYROID HORMONE EVALUATION:
- On the day of scheduled termination, each F0 animal was anesthetized with CO2/O2 and blood was collected from the vena cava or aorta
- On PND 4, trunk blood was collected from two culled pups per litter. For dams with more than two pups to be culled, blood was collected from up to two additional pups and pooled.
- On PND 13, blood was collected via intracardiac puncture from two pups per litter (one male and one female whenever possible), except for one Dam who only had one pup available at PND 13 for collection.
Oestrous cyclicity (parental animals):
Vaginal lavage samples were collected daily from F0 females for 2 weeks (Day 1 through Day 15) prior to cohabitation. The examinations continued until evidence of mating was observed. A vaginal lavage sample was collected on the day of necropsy for all F0 females, one female (control group) who did not have a sample collected. Samples were stained and examined microscopically for determination of the stage of the estrous cycle.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible), culled pups were terminated and discarded

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- external abnormalities
- number of live and dead pups of each sex
- clinical observations
- body weight was recorded on PND 0, 1, 4, 7, and 13
- Anogenital distance was measured on PND 1
- nipple/areola retention was measured for male pups on PND 13
- Collection of blood from two culled pups per litter, where possible for for possible future evaluation of thyroid hormones

GROSS EXAMINATION OF DEAD PUPS:
- yes, for external abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals on Day 31
- Maternal animals: All surviving animals on PND 14
- Animals were weighed prior to necropsy, sedated with CO2/O2, and exsanguinated.

GROSS NECROPSY
- not specified
- Tissues collected: see Table 1
- Tissue Processing: All gross lesions from all groups , all reproductive tissues from the control and high dose groups, and all preserved tissues designated for histopathologic evaluation in Table 1 from 5 animals/sex/group in the control and high dose groups

HISTOPATHOLOGY / ORGAN WEIGHTS
- 5 animals/sex/dose
- see Table 1
Postmortem examinations (offspring):
SACRIFICE/NECROPSY/HISTOPATHOLOGY:
- F1 offspring culled on PND 4 were terminated via decapitation and discarded without further examination.
- Following blood collection on PND 13, one pup/sex/litter, when available, was terminated via CO2 inhalation and the thyroid gland (with parathyroid embedded and attached to the trachea) was collected. The tissue was fixed in 10% neutral buffered formalin (NBF) for one day before the trachea was removed; the thyroid gland (with parathyroids) was weighed post-fixation and then stored in 10% NBF for possible histopathological examination.
- All remaining pups were terminated via CO2 inhalation and discarded without further examination.
Statistics:
PROVANTIS-COLLECTED DATA:
- normality was determined by the Shapiro-Wilks test
- homogeneity of variances was determined by Levene's test
- normally distributed parametric data: ANOVA F-test followed by a Dunnett's test
- not normally-distributed nonparametric data: Kruskal-Wallis test followed by a Wilcoxon tests
and the Bonferroni-Holm method
- All statistical tests were performed at the 0.05 level of significance (p < 0.05), after accounting for multiple comparisons where indicated.

FOB:
- for scoring variables and yes/no variables: chi-square test
- for variables classified as having continuous responses: one-way fixed effects analysis of variance (ANOVA), with dose group as the fixed effect
- for variables recorded as count data: square root transformation (√(value+0.5)) was applied to the data prior to fitting the one-way ANOVA model.

MOTOR ACTIVITY DATA:
- one-way parametric analysis of variance (ANOVA) with a fixed dose group effect was fitted to the measurements
- Shapiro-Wilk test for normality
- Levene’s test for constant variance
- Under the parametric one-way ANOVA approach, Dunnett’s test was performed
- Under the nonparametric approach, a Kruskal-Wallis test was applied

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test article-related clinical observations were limited to red feces observed in all animals receiving ≥ 100 mg/kg beginning on Day 2 and continuing through termination. One female in the 300 mg/kg group had a ventral mass that was no longer present at the time of scheduled termination. Incidences of abrasion, eye discharge, rough coat, thin, reduced feces, and scabbing were deemed incidental as they were limited to a single animal or were transient. Alopecia is a common clinical observation for this strain of rat and was noted in all groups. There were no abnormal maternal care observations noted during lactation.
Mortality:
no mortality observed
Description (incidence):
All animals survived until scheduled removal from study at Day 31 (males), removal for failure to deliver (GD 24), or PND 14 (delivered females).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test article-related effects on body weights and body weight changes for males. There were no test article-related effects on female body weight or body weight gains during the pre-mating period and gestation; body weights and body weight changes during the lactation period from PND 0 through 14 were comparable across groups, including control.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Test article administration did not affect male food consumption. A statistically significant increase in food consumption was noted for females in the 300 mg/kg group during the first week of study (Days 1-8). However, food consumption for this group was comparable with controls for the remainder of the study. There were no effects on food consumption for females during the gestation and lactation periods.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test article-related findings noted in the hematology, coagulation, or serum chemistry parameters. Any statistically significant or apparent differences between test article groups and control group were not considered test article related due to small magnitude of difference, absence of a dose response, and/or general overlap in magnitude of individual values with controls.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test article-related findings noted in the hematology, coagulation, or serum chemistry parameters. Any statistically significant or apparent differences between test article groups and control group were not considered test article related due to small magnitude of difference, absence of a dose response, and/or general overlap in magnitude of individual values with controls.
Endocrine findings:
no effects observed
Description (incidence and severity):
TSH and T4 concentrations were generally within one standard deviation or just outside of the zero-dose average levels indicating that the test material does not significantly affect the levels of TSH or T4 for adults (males at Day 31) or pups (PND 13).
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
The statistically significant difference seen in the home cage lethargy/arousal score for males at 100 mg/kg was not considered test article related as all scores remained within the range of normal behavior (score -1: slight lethargy; little exploration, but not prostrate (may be sleeping); 0: alert, responsive, exploratory movements; 1: slight arousal or excitement, tenseness).
There were no treatment-related differences in grip strength between all groups when compared to controls.
Reduced locomotor activity counts were observed at all dose levels for males and for females at 100 mg/kg (rearing interval counts) and 300 mg/kg; the differences were slight and not considered to be toxicologically relevant for either sex.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All F0 female uterine tissue sections from the 0 mg/kg and 1000 mg/kg dose groups had microscopic findings compatible with placental implantations.
These microscopic findings were multifocal areas of large vacuolated and pigmented macrophages in the deep myometrium. These microscopic changes are normal findings in multiparous females after parturition, and therefore, unrelated to test article exposure.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
The total number of pups born (live and dead on PND 0) was comparable at all dosages. The
litter sex ratio was comparable across all groups, including controls.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
All females were cycling normally except for one female in the 300 mg/kg group that had no completed cycles due to persistent diestrus and one female in the 1000 mg/kg group that had a single complete cycle followed by persistent diestrus. One additional 300 mg/kg female was in diestrus for more than 4 days. One 100 mg/kg female had a single cycle with 5 days in estrus. All of the females with extended estrus or diestrus mated and produced a viable litter. These cycle irregularities were not considered test article-related because of the low incidence, short duration and/or lack of effect on reproductive performance.
The mean percent days in the estrus, diestrus, and proestrus stage were similar across groups. There was a slightly increased percentage of days in diestrus for the 300 mg/kg group due to the single female in extended diestrus which lasted for more than half of the evaluation period; as a result, the percentage of days in estrus was decreased. These changes were incidental because the effects were introduced due to a single female which subsequently mated and produced offspring. There were no test article-related effects on the mean cycle length.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Reproductive parameters were not influenced by test article administration. All females had evidence of mating. One female in the 0 mg/kg group was not pregnant; at termination, no signs of implantation were observed.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test article-related clinical observations. Bruising and discolored skin, which is commonly associated with normal maternal care, was observed for a few pups in the 0, 300, and 1000 mg/kg groups. Other findings, such as damaged toes, thin, and scabbing, were not considered to be treatment-related as they were limited to a single pup within a litter and/or a single group (including controls).
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There were no decreases in viability or survival between PND 0-4 as indicated by a ≥ 98% survival index across all groups; two pups died in the 0 mg/kg group, while one pup each died in the 100, 300 and 1000 mg/kg groups during this period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Overall pup body weights, by litter, were consistent between groups. There were no test article-related differences in male or female pup body weights. Similarly, body weight changes for all pups, and when evaluated by sex, were comparable to controls.
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):
no effects observed
Description (incidence and severity):
There was no treatment-related effect on the anogenital distance for male or female pups. The mean distance was within 0.2 mm for males and 0.1 mm for females for all treated groups in comparison to the control pups.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The mean number of retained nipples/areolae for male pups was similar across all groups. There was no discernable difference in the total number of pups or litters containing at least one pup with retained nipples/areolae.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

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:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The no observable adverse effect level (NOAEL) for systemic and reproductive toxicity in F0 males and females was above 1000 mg/kg bw/day in this study. Due to the absence of toxic effects at the highed tested dose, an exact NOAEL was not determinable.
Executive summary:

The purpose of this study was to evaluate the subchronic, reproductive, and developmental toxicity of test material when administered daily via oral gavage.


Endpoints evaluated were survival, clinical observations, body weights, food consumption measurements, neurobehavioral assessments (spontaneous locomotor activity, functional observational battery, grip strength), parturition checks, litter evaluations, clinical pathology (hematology, coagulation, serum chemistry), and anatomic pathology, including organ weights, gross observations at necropsy, and microscopic evaluation of select tissues. Blood was collected for the analysis of thyroid hormone evaluation from all F0 animals and representative pups from each litter; analysis was conducted on the F0 males and on pups at postnatal day (PND) 13. Administration of the test material at dosages of 100, 300, or 1000 mg/kg to F0 male and female rats for up to 5 weeks and 8 weeks, respectively, produced no adverse effects on body weights, food consumption, reproductive performance, FOB, grip strength, clinical pathology, thyroid hormone, or anatomic pathology parameters. A decrease in rectal temperature was observed for females at 300 and 1000 mg/kg, but the relationship to test material is unclear. Reduced locomotor activity counts were observed at all dose levels for males and for females at 100 mg/kg (rearing interval counts) and 300 mg/kg; the difference was slight and not considered to be toxicologically relevant for either sex. There were no indirect treatment-related effects on male or female F1 clinical observations, body weights, anogenital distance, and thyroid hormones, or male pup areola/nipple retention.


Therefore, the no observable adverse effect level (NOAEL) for systemic and reproductive toxicity in F0 males and females was 1000 mg/kg.