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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive/developmental toxicity screening study: Read-across, NOAEL (systemic toxicity and reproductve toxicity) >= 1000 mg/kg bw/d, according OECD 422, GLP compliance, 2022, K1

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

see attached justification

Reason / purpose for cross-reference:

read-across source

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

Critical effects observed:

no

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

Reference 2

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

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

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

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

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).

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

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: 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.

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

Critical effects observed:

no

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Critical effects observed:

no

Reproductive effects observed:

no

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive/Developmental Toxicity Screening Study:

As no data for the test substance is available regarding reproduction toxicity a read-across to the category member CAS 5521-31-3 was performed.

A reproductive / developmental screening study according to OECD TG 422 and in compliance with GLP was performend 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 (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. 

 

Further toxicological data of category members:

The test article belongs to the "perylene based organic pigments" category (see attached document for details on category members and for read across justification). Regarding the reproductive toxicity, additional reliable data are available for other category members. All of the studies are taken into account for the evaluation and assessment of the toxicity of the test article.

For other category members additional screening studies according to OECD TG 422 or 421 are available. In none of the studies a evidence for reproductive toxicity was found, supporting the results of this screening study.

Effects on developmental toxicity

Description of key information

Prenatal development: Read across, NOAEL (systemic toxicity/developmental toxicity) >= 1000 mg/kg, according OECD 414, GLP compliance, 2022, K1

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

see attached justification

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Remarks:

sytsemic toxicity

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Dose descriptor:

NOAEL

Remarks:

developmental 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

Abnormalities:

no effects observed

Developmental effects observed:

no

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Aug 2021 - 22 Nov 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Wistar Hannover

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories CRL, Rhone, 327 Impasse du Domaine Les Oncines, 69210 Saint Germain Nuelles (France)
- Age at order: Males: at least 11 weeks; Females: 9 weeks
- Weight at order: Males: 325-350 g; Females: 200-225 g
- Housing: no more than 5 of one sex to a cage (before and after mating); 1 male and 1 female per cage (during mating)
- Diet: ad libitum, 4 RF 21,Mucedola S.r.l., Via G. Galilei 4, 20019 SettimoMilanese (MI), Italy
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C +/- 2 °C
- Humidity: 55 % +/- 15 %
- Air changes (per hr): approximately 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Aug 2021 To: 22 Nov 2021

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 %

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Suspensions of the test item, in 0.5 % CMC, were prepared using the following procedure:
1. the required amount of test item were weighed.
2. the required amount of vehicle was added.
3. the mixture was treated with a Silverson for 3 minutes.
4. the resulting suspension was left under magnetic stirring for at least 16 hour, at room temperature, prior to dosing and during dosing
The formulation was prepared daily at concentrations of 10, 30 and 100 mg/mL. Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): 10 mL/kg bw
- Concentration in vehicle: 0, 10, 40, 120 mg/ml
- Lot/batch no. (if required): not specified
- Purity: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated in ERBC Study no. A4346 in the range from 10 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.99; accuracy 85-115%; precision CV < 10%). A 28 hour stability at room temperature and a 9 day stability at 2-8°C (followed by one day at room temperature under magnetic stirring) were verified in the range from 10 to 100 mg/mL.
Samples of the preparations prepared on Week 1 and Last Week were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in ERBC SOPs for suspensions (85-115% for concentration and CV < 10% for homogeneity).
Chemical analysis was carried out by the Analytical Chemistry Department at ERBC.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: not specified
- Proof of pregnancy: sperm in vaginal smear or vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

from Day 6 through Day 19 post coitum (14 Days)

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Mid

Dose / conc.:

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

Remarks:

High

No. of animals per sex per dose:

25 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: given by Sponsor
- Fasting period before blood sampling for (rat) dam thyroid hormones: not specified
- Time of day for rat dam blood sampling: in the morning
- Other: on Day 20 post coitum

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twiche daily and once daily at weekends an public holidays
- Cage side observations: Check for mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: all animals, on day 0, 3, 6, 9, 12, 15, 18 and 20 post coitum

FOOD CONSUMPTION: Yes
- measured on day 0, 3, 6, 9, 12, 15, 18 and 20 post coitum
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: thyroid and brain

THYROID HORMONE DETERMINATION (T3, T4, TSH): Yes
- Blood sampled in the morning on day 20 post coitum (day of necropsy)
- slight isoflurane anaesthesia

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight
- Number of corpora lutea
- Number of implantations
- Number of intra-uterine deaths: Early resorptions (only placental remnants visible), Late resorptions (placental and foetal remnants visible)
- Number, sex and weight of all live foetuses
- Number and sex of dead foetuses (foetuses at termwithout spontaneous movements and breathing) and in each foetuses allocated to the skeletal examination
- Gross evaluation of placentae

- The uteri from females without visible implantations (one in group 1, 3, 4; five in group 2) were immersed in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation

- The uteri from dams showing unilateral implantations on the right horn (one in group 1 and 3), were also immersed in a 20% solution of ammonium sulphide, revealing the non-pregnant left horn

Blood sampling:

- Serum: Yes
- Volume: 1 mL
- in the morning of Day 20 post coitum

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
- Anogenital distance of all live rodent pups: Yes

Statistics:

For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of theWilliams test. The criterion for statistical significance was p<0.05.

Indices:

Pre-implantation loss was calculated as a percentage from the formula:

> Pre impl. Loss [%] = (no. of corpora lutea − no. of implantations) x 100/ no. of corpora lutea

Post-implantation loss was calculated as a percentage from the formula:

> Post impl. Loss [%] = (no. of implantations − no. of live foetuses) x 100/ no. of implantations

Total implantation loss was calculated as a percentage from the formula:

> Total impl. Loss [%] = (no. of corpora lutea − no. of live foetuses) x 100/ no. of corpora lutea

Sex ratios of the foetuses were calculated as the percentage of males.

All derived values (e.g., means, percentages, ratios) first were calculated within the litter and the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters.

Historical control data:

Historical control data from 2014 to 2022 was provided. Data included: foetal external abnormalities, skeletal examination, fixed visceral examination, fate of females, macroscopic observation of females at final cesarean section, litter data and sex ratio of dams with live foetuses at necropsy. Tables were extracted from reproductive toxicology historical control data.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Hairloss was observed in one control, two mid-dose and one high dose females. Considering the low incidence of hairloss and the presence of the sign also in a control animal the observation was deemed representative of normal background variability within the Wistar Han rat.
During the treatment period a presence of couloured faeces (red) in all treated group. Considering that the test item is a red solid the colour indicated the presence of the substance in the faeces and not an abnormalities.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and weight gain were unaffected in females treated up to 1000 mg/kg/day over the entire administration period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The statistically significant increase in food consumption (29.7 g of high dose group versus 27.4 g of controls) was considered incidental and unrelated to treatment.

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

Endocrine findings:

no effects observed

Description (incidence and severity):

No changes were observed in the determination of T3, T4 and TSH.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Uterus weight, corrected maternal body weight and weight gain were not affected by treatment.
There were no treatment-related organ weight changes (brain and thyroid gland) at the end of the treatment period. Any variations were considered to be within the range of expected spontaneous changes in rats of the same age and unrelated to treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related macroscopic observations at the end of the treatment period. Any macroscopic observations were within the range of occasionally observed and expected spontaneous changes in rats of the same age and therefore considered unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related microscopic observations in the thyroid gland at the end of the treatment period. Any microscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Litter data of treated females were comparable to controls.
Presence of foetuses with a weight less that 2.7 g and classified as small were noted in control, mid and high dose groups. Considering that the incidence of the high dose group is lower than controls and in the absence of the dose relation trend the presence is consider incidental and unrelated to treatment.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One foetus with malformation was observed in the mid- dose group. The foetus showed unilateral fusion of thoracic arches no. 10th and 11th in combination with the fusion of the 10th and 11th ribs. Considering that no other observations were noted related to the thoracic arches or ribs and in the absence of the relationship the changes were considered spontaneous in origin.
The other changes noted were comparable between the control and the treated groups.

Visceral malformations:

no effects observed

Description (incidence and severity):

The variations observed occurred with similar incidence across all groups including controls.

Dose descriptor:

NOAEL

Remarks:

developmental 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

Abnormalities:

no effects observed

Developmental effects observed:

no

 

 

Table 1: CLINICAL SIGNS OF FEMALES – GROUP INCIDENCE
Interval: 0 - 20 Days Group	1		2		3		4
Observation	(25)		(25)		(25)		(25)
APPEARANCE	a	b	a	b	a	b	a	b
Hairloss	1	4.0	0	0.0	2	8.0	1	4.0
() = Number of animals alive at start of interval a = Number of animals affected b = Percent of animals with observation during interval

 

 

Table 2: ABSOLUTE ORGAN WEIGHTS (g) - GROUP MEAN DATA
Organ: Brain	Data homogeneous by Bartlett's test (Dunnett's test)
Group	Control (Group 1)	2	3	4
Number/group	25	25	25	25
Mean	1.853	1.848	1.860	1.851
Standard deviation	0.073	0.066	0.091	0.070
Group diff. at p < 0.05		0.051	0.051	0.051
Group diff. at p < 0.01		0.064	0.064	0.064
Analysis of variance: F ratio = 0.12; Df = 3/ 96; F probability = 0.942 Note: a * indicates group mean is significantly different from control at level of significance shown.
Organ: Thyroid	Data homogeneous by Bartlett's test (Dunnett's test)
Group	Control (Group 1)	2	3	4
Number/group	25	25	25	25
Mean	0.0218	0.0236	0.0219	0.0232
Standard deviation	0.0048	0.0037	0.0045	0.0044
Group diff. at p < 0.05		0.0030	0.0030	0.0030
Group diff. at p < 0.01		0.0037	0.0037	0.0037
Analysis of variance: F ratio = 1.09; Df = 3/ 96; F probability = 0.358 Note: a * indicates group mean is significantly different from control at level of significance shown.

 

 

Table 3: ORGAN WEIGHTS° TO BRAIN WEIGHT - GROUP MEAN DATA
Organ: Thyroid	Data homogeneous by Bartlett's test (Dunnett's test)
Group	Control (Group 1)	2	3	4
Number/group	25	25	25	25
Mean	1.181	1.280	1.181	1.256
Standard deviation	0.268	0.198	0.254	0.240
Group diff. at p < 0.05		0.163	0.163	0.163
Group diff. at p < 0.01		0.204	0.204	0.204
Analysis of variance: F ratio = 1.11; Df = 3/ 96; F probability = 0.349 Note: a * indicates group mean is significantly different from control at level of significance shown. ° = expressed as % organ to brain weight ratio

 

 

Table 4: MACROSCOPIC OBSERVATIONS OF FEMALES – FINAL SACRIFICE - GROUP INCIDENCE
Group	1	2	3	4
Number in group	25	25	25	25
Caecum
-        Abnormal contents	0	0	0	1
Forelimbs
-        Hairloss	1	0	1	1
Uterus
-        Unilateral implantation	1	0	1	0
-        Not pregnant	1	4	1	1
-        Total resorption	0	1	0	0
Whole animal
-        No abnormalities detected	22	20	22	23

 

 

Table 5: EXTERNAL EXAMINATION OF FOETUSES - GROUP INCIDENCE
Group	Organ	Cat	Observation(s)	No. Observed	Foetuses Affected	%	No.             Observed	Litters Affected	%
1	Whole foetus		No abnormalities detected	285	280	98.25	24	-	-
	Whole foetus	AN	Small	285	5	1.75	24	4	16.67
2	Whole foetus		No abnormalities detected	228	228	100.00	20	20	100.00
3	Whole foetus		No abnormalities detected	272	265	97.43	24	-	-
	Whole foetus	AN	Small	272	7	2.57	24	5	20.83
4	Whole foetus		No abnormalities detected	290	289	99.66	24	-	-
	Whole foetus	AN	Small	290	1	0.34	24	1	4.17

 

 

 

Table 6: SKELETAL EXAMINATION OF FOETUSES - GROUP INCIDENCE
				No. Foetuses			No. Litters
Group	Organ	Cat	Observation(s)	Obs	Aff	%	Obs	Aff	%
1	Forepaw(s)	AN	Metacarpal(s) no ossification 4th	150	70	46.67	24	23	95.83
	Lumbar vertebrae	VA	Centrum incomplete ossification	150	1	0.67	24	1	4.17
	Pelvic girdle	AN	Pubis incomplete ossification	150	1	0.67	24	1	4.17
	Ribs	AN	Wavy	150	10	6.67	24	8	33.33
	Ribs	VA	Short 14th	150	65	43.33	24	22	91.67
	Ribs	VA	Rudimentary 14th	150	11	7.33	24	9	37.50
	Ribs	VA	14 ribs	150	15	10.00	24	9	37.50
	Sacral vertebrae	AN	Arch(es) incomplete ossification	150	8	5.33	24	3	12.50
	Skull	AN	Hyoid no ossification	150	4	2.67	24	3	12.50
	Skull	AN	Temporal incomplete ossification	150	16	10.67	24	10	41.67
	Skull	AN	Frontal incomplete ossification	150	1	0.67	24	1	4.17
	Skull	AN	General incomplete ossification	150	5	3.33	24	4	16.67
	Skull	VA	Supraoccipital incomplete ossification	150	60	40.00	24	20	83.33
	Skull	VA	Interparietal incomplete ossification	150	41	27.33	24	17	70.83
	Skull	VA	Parietal incomplete ossification	150	28	18.67	24	15	62.50
	Sternebrae	AN	Fused	150	1	0.67	24	1	4.17
	Sternebrae	AN	No ossification	150	2	1.33	24	2	8.33
	Sternebrae	VA	No ossification 5th	150	7	4.67	24	6	25.00
	Sternebrae	VA	Incomplete ossification 5th	150	22	14.67	24	11	45.83
	Sternebrae	VA	Incomplete ossification 6th	150	43	28.67	24	17	70.83
	Thoracic vertebrae	AN	Centrum bipartite and asymmetical	150	2	1.33	24	2	8.33
	Thoracic vertebrae	VA	Centrum incomplete ossification	150	6	4.00	24	3	12.50
	Thoracic vertebrae	VA	Centrum dumb-bell shaped	150	1	0.67	24	1	4.17
2	Forepaw(s)	AN	Metacarpal(s) incomplete ossification	119	1	0.84	20	1	5.00
	Forepaw(s)	AN	Metacarpal(s) no ossification 4th	119	37	31.09	20	15	75.00
	Ribs	AN	Wavy	119	9	7.56	20	6	30.00
	Ribs	VA	14 ribs	119	10	8.40	20	6	30.00
	Ribs	VA	Short 14th	119	52	43.70	20	18	90.00
	Ribs	VA	Rudimentary 14th	119	12	10.08	20	11	55.00
	Sacral vertebrae	AN	Arch(es) incomplete ossification	119	1	0.84	20	1	5.00
	Skull	AN	Frontal incomplete ossification	119	1	0.84	20	1	5.00
	Skull	AN	Temporal incomplete ossification	119	6	5.04	20	4	20.00
	Skull	AN	Hyoid no ossification	119	1	0.84	20	1	5.00
	Skull	VA	Interparietal incomplete ossification	119	26	21.85	20	11	55.00
	Skull	VA	Parietal incomplete ossification	119	11	9.24	20	8	40.00
	Skull	VA	Supraoccipital incomplete ossification	119	42	35.29	20	19	95.00
	Sternebrae	VA	Incomplete ossification	119	1	0.84	20	1	5.00
	Sternebrae	VA	Incomplete ossification 6th	119	21	17.65	20	10	50.00
	Sternebrae	VA	No ossification 5th	119	3	2.52	20	3	15.00
	Sternebrae	VA	Incomplete ossification 5th	119	20	16.81	20	11	55.00
	Thoracic vertebrae	AN	Centrum bipartite	119	1	0.84	20	1	5.00
	Thoracic vertebrae	AN	Centrum bipartite and asymmetrical	119	1	0.84	20	1	5.00
	Thoracic vertebrae	VA	Centrum asymmetrical dumb-bell shaped	119	1	0.84	20	1	5.00
	Thoracic vertebrae	VA	Centrum dumb-bell shaped	119	1	0.84	20	1	5.00
	Thoracic vertebrae	VA	Centrum incomplete ossification	119	4	3.36	20	2	10.00
3	Forepaw(s)	AN	Metacarpal(s) incomplete ossification	144	4	2.78	24	1	4.17
	Forepaw(s)	AN	Metacarpal(s) no ossification 4th	144	65	45.14	24	20	83.33
	Ribs	AN	Wavy	144	7	4.86	24	5	20.83
	Ribs	MA	Fused	144	1	0.69	24	1	4.17
	Ribs	VA	14 ribs	144	16	11.11	24	10	41.67
	Ribs	VA	Rudimentary 14th	144	11	7.64	24	9	37.50
	Ribs	VA	Short 14th	144	78	54.17	24	22	91.67
	Sacral vertebrae	AN	Arch(es) incomplete ossification	144	5	3.47	24	3	12.50
	Skull	AN	Frontal incomplete ossification	144	1	0.69	24	1	4.17
	Skull	AN	Hyoid no ossification	144	4	2.78	24	1	4.17
	Skull	AN	General incomplete ossification	144	2	1.39	24	2	8.33
	Skull	AN	Temporal incomplete ossification	144	14	9.72	24	6	25.00
	Skull	VA	Parietal incomplete ossification	144	28	19.44	24	12	50.00
	Skull	VA	Interparietal incomplete ossification	144	36	25.00	24	14	58.33
	Skull	VA	Supraoccipital incomplete ossification	144	58	40.28	24	21	87.50
	Sternebrae	AN	Bipartite 5th	144	1	0.69	24	1	4.17
	Sternebrae	AN	No ossification	144	1	0.69	24	1	4.17
	Sternebrae	VA	Incomplete ossification	144	5	3.47	24	3	12.50
	Sternebrae	VA	Incomplete ossification 6th	144	36	25.00	24	13	54.17
	Sternebrae	VA	No ossification 5th	144	10	6.94	24	5	20.83
	Sternebrae	VA	Incomplete ossification 5th	144	22	15.28	24	16	66.67
	Thoracic vertebrae	AN	Centrum bipartite and asymmetrical	144	2	1.39	24	2	8.33
	Thoracic vertebrae	AN	Centrum asymmetrical ossification	144	1	0.69	24	1	4.17
	Thoracic vertebrae	AN	Centrum bipartite	144	1	0.69	24	1	4.17
	Thoracic vertebrae	MA	Arch(es) fused	144	1	0.69	24	1	4.17
	Thoracic vertebrae	VA	Centrum asymmetrical dumb-bell shaped	144	1	0.69	24	1	4.17
	Thoracic vertebrae	VA	Centrum incomplete ossification	144	1	0.69	24	1	4.17
4	Forepaw(s)	AN	Metacarpal(s) no ossification 4th	150	52	34.67	24	19	79.17
	Ribs	AN	Wavy	150	12	8.00	24	6	25.00
	Ribs	VA	Rudimentary 14th	150	6	4.00	24	6	25.00
	Ribs	VA	14 ribs	150	11	7.33	24	8	33.33
	Ribs	VA	Short 14th	150	71	47.33	24	21	87.50
	Sacral vertebrae	AN	Arch(es) incomplete ossification	150	5	3.33	24	4	16.67
	Skull	AN	General incomplete ossification	150	2	1.33	24	2	8.33
	Skull	AN	Frontal incomplete ossification	150	1	0.67	24	1	4.17
	Skull	AN	Hyoid no ossification	150	4	2.67	24	4	16.67
	Skull	AN	Temporal incomplete ossification	150	17	11.33	24	9	37.50
	Skull	VA	Interparietal incomplete ossification	150	46	30.67	24	18	75.00
	Skull	VA	Supraoccipital incomplete ossification	150	60	40.00	24	19	79.17
	Skull	VA	Parietal incomplete ossification	150	30	20.00	24	14	58.33
	Sternebrae	AN	Asymmetrical ossification 5th	150	1	0.67	24	1	4.17
	Sternebrae	VA	Incomplete ossification 5th	150	32	21.33	24	17	70.83
	Sternebrae	VA	No ossification 5th	150	4	2.67	24	3	12.50
	Sternebrae	VA	Incomplete ossification	150	1	0.67	24	1	4.17
	Sternebrae	VA	Incomplete ossification 6th	150	33	22.00	24	13	54.17
	Thoracic vertebrae	AN	Centrum bipartite	150	1	0.67	24	1	4.17
	Thoracic vertebrae	VA	Centrum asymmetrical dumb-bell shaped	150	1	0.67	24	1	4.17
	Thoracic vertebrae	VA	Centrum incomplete ossification	150	4	2.67	24	4	16.67

Conclusions:

On the basis of the results, the dosage of >=1000 mg/kg/day is considered the NOAEL for maternal and embryo-foetal development.

Executive summary:

The effects of the test material were investigated, in female Wistar Hannover rats during pregnancy and embryo-foetal development, from gestation Day 6 through Day 19 in a GLP compliant study according to OECD TG 414. 

Females were mated with sexually mature males of the same strain and then assigned to 4 groups of 25 females each. The plan for this study was to investigate doses of 100, 300 and 1000 mg/kg bw/day of the test item with a dose volume of 10 mL/kg body weight, during the gestation period from Day 6 through Day 19 post coitum. Control females received the vehicle (0.5 % carboxymethyl cellulose (CMC)) at the same dose volume during the same treatment period.
Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination. Thyroid hormone determination was performed. The brain and thyroid were weighed. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, gravid uterus weights, foetal weight and sex were recorded. All foetuses were examined for external abnormalities. The anogenital distance (AGD) in all live foetuses was recorded. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.

All females survived until scheduled necropsy. No signs of discomfort or clinical symptoms from the treatment with the test item were observed. No macroscopic findings were noted during necropsy of the females. Mean food consumption, mean body weight and corrected body weight gain (corrected for the gravid uterus weight) were not affected by treatment with the test item in any dose group. Post-implantation losses and the mean number of foetuses per dam were not affected by treatment with the test item at all dose levels. No test item-related effects on foetal body weights were noted. No test item-related effects on foetal sex ratios or anogenital distance were noted in any dose group.
During the external examination of the foetuses, no test item-related abnormal findings were noted. Hormone levels of dams were comparable between groups. Females did not show any macroscopic or microscopic (thyroid) changes related to treatment. Skeletal and visceral examinations were comparable between groups.

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

Prenatal Developmental Toxicity:

As no data for the test substance is available regarding prenatal developmental toxicity a read-across to the category member CAS 5521-31-3 was performed.

Females were mated with sexually mature males of the same strain and then assigned to 4 groups of 25 females each. The plan for this study was to investigate doses of 100, 300 and 1000 mg/kg bw/day of the test item with a dose volume of 10 mL/kg body weight, during the gestation period from Day 6 through Day 19 post coitum. Control females received the vehicle (0.5 % carboxymethyl cellulose (CMC)) at the same dose volume during the same treatment period.
Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination. Thyroid hormone determination was performed. The brain and thyroid were weighed. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, gravid uterus weights, foetal weight and sex were recorded. All foetuses were examined for external abnormalities. The anogenital distance (AGD) in all live foetuses was recorded. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.

All females survived until scheduled necropsy. No signs of discomfort or clinical symptoms from the treatment with the test item were observed. No macroscopic findings were noted during necropsy of the females. Mean food consumption, mean body weight and corrected body weight gain (corrected for the gravid uterus weight) were not affected by treatment with the test item in any dose group. Post-implantation losses and the mean number of foetuses per dam were not affected by treatment with the test item at all dose levels. No test item-related effects on foetal body weights were noted. No test item-related effects on foetal sex ratios or anogenital distance were noted in any dose group.
During the external examination of the foetuses, no test item-related abnormal findings were noted. Hormone levels of dams were comparable between groups. Females did not show any macroscopic or microscopic (thyroid) changes related to treatment. Skeletal and visceral examinations were comparable between groups.

 

Further toxicological data of category members:

The test article belongs to the "perylene based organic pigments" category (see attached document for details on category members and for read across justification). Regarding the developmental toxicity, additional reliable data are available for other category members. All of the studies are taken into account for the evaluation and assessment of the toxicity of the test article.

For other category members additional screening studies according to OECD TG 422 or 421 are available. In none of the studies a evidence for reproductive or developmental toxicity was found, supporting the results of this read-across study.

Justification for classification or non-classification

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

The available screening study is reliable and suitable for classification purposes under Regulation 1272/2008. No adverse effects on fertility or development were observed in a screening study in rats (OECD 422) and no adverse effects were observed in a teratogenicity/developmental toxicity study in rats (OECD 414). 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 fourteenth time in Regulation (EC) No. 2020/217.

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