2,9-bis(p-methoxybenzyl)anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Sep 2021 - 27 Dec 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

Test material information

Constituent 1

Reference substance name:

2,9-bis(p-methoxybenzyl)anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone

EC Number:

280-472-4

EC Name:

2,9-bis(p-methoxybenzyl)anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone

Cas Number:

83524-75-8

Molecular formula:

C40H26N2O6

IUPAC Name:

7,18-bis[(4-methoxyphenyl)methyl]-7,18-diazaheptacyclo[14.6.2.2²,⁵.0³,¹².0⁴,⁹.0¹³,²³.0²⁰,²⁴]hexacosa-1(23),2,4,9,11,13,15,20(24),21,25-decaene-6,8,17,19-tetrone

Test material form:

solid: nanoform, no surface treatment

Details on test material:

- State of aggregation: solid, powder
- Particle size distribution (TEM): 82.9 nm (D50)
- Mass median aerodynamic diameter (MMAD): not specified
- Geometric standard deviation (GSD): not specified
- Shape of particles: elongated, rod
- Surface area of particles: 37 m²/g
- Crystal structure: crystalline
- Coating: no
- Surface properties: not applicable
- Density: 1470 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) approx. 12 wks
- Weight at study initiation: (P) Males: 334.9 - 452.1 g; Females: 241.9 - 273.8 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: 25 d

ENVIRONMENTAL CONDITIONS
- Temperature: 70 to 74 °F (ca. 21.1 - 23.3 °C)
- Humidity: 43 to 66 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: September 28, 2021 To: December 27, 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): 5.0 mL/kg (group 1, 2, 3); 5.7 mL/kg (group 4)
- 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 September 20, 2021, stored at room temperature and refrigerated, 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.

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 / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control (Group 1)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Remarks:

Low (Group 2)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Remarks:

Mid (Group 3)

Doses / concentrations 4

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: at least once daily

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, or until the 14-day cohabitation period was completed. A vaginal lavage sample was collected on the day of necropsy for all F0 females, except 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 black feces observed in all animals receiving ≥ 100 mg/kg beginning as early as Day 2 and continuing through termination. One female in the 0, 300, and 1000 mg/kg groups and two females in the 100 mg/kg group had a ventral mass develop during gestation. With the exception of the 300 mg/kg female, the mass was no longer present at the time of scheduled termination; this was not considered to be test article-related as the incidence rate was similar across all groups including controls. Occurrences of rales, eye discharge, mouth discharge, and scabbing were deemed incidental as they were limited to a single animal and did not persist. Alopecia is a common clinical observation for this strain of rat and was noted in all groups. One female in the 100 mg/kg group displayed reduced maternal care which resulted in the loss of the entire litter by PND 1.

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 significantly higher food consumption was noted for females receiving ≥100 mg/kg group during the first week of study (Days 1-8) and for females in the 100 mg/kg group from GD 7-14. Increases in food consumption were not considered to be related to test article administration as they were not dose-dependent, and consumption was comparable with controls for the remainder of the study periods, including lactation.

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):

A statistically significant difference in home cage posture for males receiving ≥ 100 mg/kg was attributed to test article administration because the difference was due to one 0 mg/kg animal with a score of 0 (awake, alert, without rearing) while all other animals had a score of -1 (sleeping, resting, recumbent). Awake or alert and sleeping or resting are normal rodent home cage behaviors.
A statistically significantly higher number of lines crossed when assessed in the open field was noted for the 1000 mg/kg females. The difference from controls was slight, and there were no corroborating effects on locomotor activity observed; therefore, the difference was not considered to be test article-related.
There were no treatment-related differences in grip strength between all groups when compared to controls.
There were no toxicologically relevant effects on locomotor activity.
Some statistically significant differences for females when analyzed per 5-minute interval. As there were no corresponding differences in total session counts, and/or some differences were attributed to high individual variability, the differences in interval counts from controls were not considered to be test article-related or toxicologically relevant.
One male (300 mg/kg) had an unusually high number of rearing counts in the 55-60 minute interval, which contributed to a very high standard error for this group within the interval and for habituation. One female (300 mg/kg) had much lower locomotor activity counts for all endpoints than all other animals in the group, which contributed to overall lower mean scores. The observed individual variability was considered to be incidental in nature.

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):

One male (1000 mg/kg) was observed with bilateral small (0.5×) epididymides and testes. Small epididymides correlated with bilateral marked ductal atrophy microscopically whereas small testes correlated with bilateral marked atrophy of the germinal epithelium. As this was noted in only one male and are known to be a spontaneous lesion in Sprague Dawley rats, this was considered an incidental finding. However, atrophy of the epididymal ducts was associated with a lack of sperm; as a result, the male was unable to sire a litter.
All F0 female uterine tissue sections from the 0 mg/kg and 1000 mg/kg dose groups had microscopic findings compatible with placental implantations; the uterus of one female (0 mg/kg) was not examined. 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:

effects observed, non-treatment-related

Description (incidence and severity):

A mammary mass was observed for one female (300 mg/kg) corresponding to a fibroadenoma microscopically. Fibroadenomas are common spontaneous mammary tumor commonly seen in Sprague Dawley rats and, therefore, not considered to be related to test material exposure.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no test article-related effects on the mean cycle length, number of cycles, or length of estrus. All females were cycling normally including the females that failed to produce a litter.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Reproductive parameters were not influenced by test article administration. There was no test article-related difference in the precoital interval or gestation length.
The female mating index was 100% for the 0, 100, and 300 mg/kg groups and 80% for the 1000 mg/kg group. However, one female successfully delivered a litter although evidence of mating was not observed. When adjusted, the female mating index was 90% for the 1000 mg/kg group. The failure of a second female to produce a litter was related to infertility of the male. As such, the female mating index was comparable between all groups.
Test article administration had no effect on the female fertility index. The female fertility index was 80%, 100%, 90%, and 100% for the 0, 100, 300, and 1000 mg/kg groups, respectively. At termination, no signs of implantation were observed for the two females in the 0 mg/kg group and one female from the 300 mg/kg group.

Effect levels (P0)

1

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

2

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

Target system / organ toxicity (P0)

Critical effects observed:

no

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 ≥ 95% survival index across all groups; six pups in the 100 mg/kg group and four pups in the 300 mg/kg group died during this period. Within the 100 mg/kg group, six pups were dead on PND 0 and five pups were dead on PND 1 for one female (100 mg/kg), resulting in a whole litter loss.

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 0.1 mm for males and 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

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.

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 the 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 (FOB), 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 test material at dosages of 100, 300, or 1000 mg/kg to F₀ male and female rats for up to 5 weeks and 9 weeks, respectively, produced no adverse effects on body weights, food consumption, estrous cyclicity, reproductive performance, FOB, grip strength, clinical pathology, thyroid hormone, or anatomic pathology parameters.

There were no 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.