Glycerol, propoxylated, esters with acrylic acid

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

The substance was tested in a Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD TG 422 and in compliance with GLP (ReachCentrum, 2019A). Wistar Han rats were treated by daily oral gavage at dose levels of 150, 375 and 750 mg/kg bw/day. No treatment-related changes were noted in any of the reproductive parameters investigated in this study. No developmental toxicity was observed up to the highest dose level tested (750 mg/kg bw/day). No toxicologically significant changes were noted in any of the developmental parameters investigated in this study.

 

Parental (systemic) NOAEL: 375 mg/kg bw/day, based on treatment related mortality at 750 mg/kg bw/day.

Parental (local) NOAEL: 150 mg/kg bw/day, based on adverse local forestomach effects at 375 mg/kg bw/day (males) and 750 mg/kg bw/day (both sexes).

Reproduction NOAEL: at least 750 mg/kg bw/day.

Developmental NOAEL: at least 750 mg/kg bw/day.

 

Based on the results of the extended one generation reproductive toxicity study (ReachCentrum, 2019C) with the read-across substance (CAS 42978-66-5) the following no-observed-adverse-effect levels (NOAEL) were established for the read-across substance:

General toxicity (F0 and F1): at least 100 mg/kg bw/day

Reproduction: at least 100 mg/kg bw/day

Developmental: at least 100 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Jun 2018 - 20 Dec 2018

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:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: males were 10-11 weeks old, females were 13-14 weeks old
- Weight at study initiation: males weighed between 262 and 333 g, females weighed between 195 and 239 g
- Housing: up to 5 animals of the same sex and same dosing group together in polycarbonate cages (Macrolon, MIV type)
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages,MIV type) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type). Pups were housed with the dam.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage cageenrichment, bedding material, food and water.
- Diet: ad libitum, Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum, Municipal tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22
- Humidity (%): 43 to 73
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at room temperature in daily portions up to a maximum of eight days before use. If not used on the day of preparation, the formulations were stored in the refrigerator. On the day of use, the dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. All formulations were used for dosing within 6 hours after preparation, if used on the day of preparation, or within 6 hours after removal from the refrigerator.
No correction was made for the purity/composition of the test item. Adjustment was made for specific gravity of the vehicle and test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was selected as vehicle, at request of the Sponsor and based on previously performed studies.
- Concentration in vehicle: 5 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- Once mating had occurred, the males and females were separated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Males were treated for 29 days, i.e. 14 days prior to mating, during mating and up to and including the day before scheduled necropsy. Females that delivered were treated for 50-58 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 39-54 days.

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

375 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 14-day dose range finder with oral administration of the test item in rats, and in an attempt to produce graded responses to the test item.

- Fasting period before blood sampling for clinical biochemistry: yes

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION: Yes
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION AND COMPOUND INTAKE: No data
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was expected or noted at visual inspection of the water bottles.

FUNCTIONAL TESTS
Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 9-12). These tests were performed after dosing, after completion of clinical observations and arena observations, if applicable. The following tests were performed: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength, Locomotor activity

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.

Sperm parameters (parental animals):

Parameters examined in all male parental generations:
testis weight, epididymis weight, spermatogenic profiling

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); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:

Clinical Observations: Clinical observations were performed at least once daily for all pups. Pups were observed daily for general health/mortality. The number of live and dead pups was determined on PND 1 and daily thereafter.
Body Weights: Live pups were weighed individually on PND 1, 4, 7 and 13.
Anogenital Distance: Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
Areola/Nipple Retention: All male pups in each litter were examined for the number of areola/nipples on PND 13.

GROSS EXAMINATION OF DEAD PUPS:
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally). Pups found dead during the weekend were fixed in identified containers containing 70% ethanol as they were not necropsied on the same day. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
Scheduled necropsies were conducted on the following days:
Males (which sired and failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
Females which delivered: PND 14-16.
Females which failed to deliver: With evidence of mating: Post-coitum Day 26 (nos. 41, 49, 58, 72).
Without evidence of mating: 26 days after the last day of the mating period (no. 46).
Females with total litter loss: Dams with no surviving pups were euthanized within 24 hours after the last pup was found dead or missing.

All males surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0-females were not fasted.

GROSS NECROPSY
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
see table 1 and 2

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day of necropsy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: only males
- How many animals: 5/sex/group
- Parameters checked: White blood cells (WBC), Neutrophil (absolute), Lymphocyte (absolute), Monocyte (absolute), Eosinophil (absolute), Basophil (absolute), Red blood cells, Reticulocyte (absolute), Red Blood Cell Distribution Width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet, Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)

Blood samples were processed for serum, and serum was analyzed for total Thyroxine (T4). Measurement of total T4 was conducted for F0-males. For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH;both sexes) was considered not relevant because no treatment-related changes in T4 were noted in F0-males, no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight were recorded.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of necropsy
- Animals fasted: only males
- How many animals: 5/sex/group
- Parameters checked: Alanine aminotransferase (ALAT), Glucose, Aspartate aminotransferase (ASAT), Cholesterol, Alkaline Phosphatase (ALP), Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Urea, Calcium, Creatinine, Inorganic Phosphate (Inorg. Phos)

Postmortem examinations (offspring):

SACRIFICE
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 7-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol®
20%). The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

GROSS NECROPSY
Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.

On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by decapitation, between 7.00 and 10.30 a.m., and samples were pooled to one sample per litter. If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much blood as possible was collected from this single pup. On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female). Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16.

Statistics:

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

see table 5

Offspring viability indices:

see table 5

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related clinical signs were noted during daily detailed clinical observations from 150 mg/kg bw/day.
Salivation was seen after dosing among animals of the 150, 375 and 750 mg/kg dose group in a dose related trend. At 150 mg/kg, salivation was noted from Day 6 onwards in most animals and this lasted until the end of treatment in males and until Day 33 of treatment in females. At 375 and 750 mg/kg, salivation was generally noted from start treatment onwards throughout the entire treatment period.
Incidental findings that were noted included scabs in the neck, rales, alopecia and red discharge of the eye (chromodacryorrhoea). These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were not considered to be signs of toxicological relevance.

Mortality:

mortality observed, treatment-related

Description (incidence):

Treatment related mortality occurred at 750 mg/kg bw/day. Two males and one female were sacrificed on Day 6, Day 27 and Day 30 of treatment, respectively. For one male and one female, test item-related findings in the forestomach were
considered to be the main cause of moribundity. For the other male the cause of mortality could not be established.
At 375 mg/kg, one female was found dead on Day 13 of treatment. Apart from salivation, no clinical symptoms were observed during the days prior to the death of this female. At macroscopic examination the lungs were not collapsed, an irregular surface of the forestomach was found and the thymus was gelatinous. No cause of death was established due to severe autolysis of the intestinal organs. As this concerned a single animal at 375 mg/kg bw/day, this was considered to be a chance finding. Females nos. 56 (150 mg/kg bw/day) and 67 (375 mg/kg bw/day) were euthanized on Lactation Day 3 and 1, respectively, as they had a total litter loss.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in body weight or body weight gain were recorded up to 750 mg/kg bw/day. Mean body weight gain of males at 750 mg/kg was reduced from Day 8 of treatment onwards, reaching statistical significance during the mating period. As resulting differences in mean body weight remained minimal (1-4%) when compared with concurrent control, this decrease was considered not to be toxicologically relevant. The statistical significant change in females at 750 mg/kg at Day 1 of treatment was considered to be unrelated to treatment as these measurements were performed prior to their first dose.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after correction for body weight were recorded up to 750 mg/kg bw/day. Absolute and relative mean food consumption of males at 750 mg/kg was reduced when
compared with concurrent control during the first week of treatment. As no statistical significance was achieved and food consumption recovered to normal levels in subsequent intervals, this temporary decrease in food consumption was considered not toxicologically relevant.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase was noted in the mean neutrophil value of males at 750 mg/kg bw/day. The mean value was approximately 2 times higher when compared with concurrent controls.
The apparent treatment-related increase noted in mean value of reticulocytes of treated females was attributed to a slightly low mean control value combined with a single female at 750 mg/kg with a value above the upper limit of historical control data (no. 76) 4. As such and as no statistical significance was achieved, the increase was considered not to be toxicologically relevant. The decrease in mean corpuscular volume (MCV) of approximately 6% compared with concurrent controls in females at 375 mg/kg was considered unrelated to administration of the test item due to the minimal magnitude of the change and absence of a dose response. Coagulation parameters of treated rats were considered not to have been affected by
treatment. The statistical significant decreases in prothrombin time noted in males at 150 and 750 mg/kg bw/day of 8 and 6%, respectively, were considered unrelated to administration of the test item due to the minimal magnitude of the change and absence of a dose response.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted in clinical chemistry parameters up to 750 mg/kg bw/day.
Mean bile acid concentration was 1.6x and 2.3x higher in females at 375 and 750 mg/kg, respectively, as concurrent control mean. No statistical significance was achieved and as this was mainly caused by two females with extremely high values (nos. 66, 375 mg/kg and 77, 750 mg/kg), values of remaining females remained within normal limits, the increase at 375 and 750 mg/kg was considered not toxicologically relevant. At 750 mg/kg, albumin levels were increased in males with 5%, and for males at 150 and 750 mg/kg, glucose levels were decreased with 20 and 23%, respectively. The alterations in clinical biochemistry parameters were considered unrelated to administration of the test item due to the minimal magnitude of the change (albumin) and absence of a dose response. In addition, mean control glucose levels were relatively high when compared with historical control data.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose males. In females, mean grip strength of the fore legs was increased with 9 and 16% at 375 and
750 mg/kg bw/day, respectively, when compared with concurrent control. In addition, mean grip strength of the hind legs was increased with 7 and 13% in females at 375 and 750 mg/kg, respectively. No statistical significance was achieved and as all values remained within historical control data, these differences were considered not to be toxicologically relevant. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment were noted in the stomach of both sexes, in males starting at 150 mg/kg bw/day; in females starting at 375 mg/kg bw/day, in the liver of males at 750 mg/kg bw/day and the spleen of females at 750 mg/kg bw/day. A combination of microscopic findings was recorded for the forestomach and consisted of ulcers and/or lymphogranulocytic inflammation and/or squamous cell hyperplasia and/or hyperkeratosis and/or edema in the submucosa of the forestomach, with in some animals newly formed blood vessels in the submucosa. At 150 mg/kg, microscopic findings were recorded in 3/6 males and consisted of lymphogranulocytic inflammation (minimal) and/or squamous cell hyperplasia (minimalslight) and/or edema (minimal-slight). At 375 mg/kg, microscopic findings were recorded in 10/10 males and 4/9 females and consisted of ulcers (males: minimal) and/or lymphogranulocytic inflammation (minimalslight)
and/or squamous cell hyperplasia (minimal-moderate) and/or hyperkeratosis (minimalmoderate) and/or edema (minimal-moderate). At 750 mg/kg, microscopic findings were recorded in 10/10 males and 10/10 females and consisted of ulcers (males: minimal-marked, females: minimal) and/or lymphogranulocytic inflammation (minimal-moderate) and/or squamous cell hyperplasia (minimal-marked) and/or hyperkeratosis (slight-marked) and/or edema (minimal-moderate).
The squamous cell hyperplasia and/or hyperkeratosis of the forestomach was considered to be the main microscopic correlate to the irregular surface of the forestomach recorded at necropsy.
In the liver of males minimal centrilobular hepatocellular hypertrophy was recorded in 2/5 males at 750 mg/kg at the end of the scheduled treatment period. This was the microscopic correlate to the weight increase recorded for males at 750 mg/kg.
In the spleen of females an increased incidence and severity of extramedullary hematopoiesis was recorded at 750 mg/kg. In the females of the scheduled sacrifice, necropsied on PND 14-16, this was recorded in 5/5 females at minimal to slight degree, compared to a minimal degree of extramedullary hematopoiesis in the spleen in 3/5 control females, 1/5 females at 150 mg/kg/day and 2/5 females at 375 mg/kg, which is considered to be within background.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone analyses:
Mean T4 values for males were slightly increased in an apparent dose-related trend. However, as no statistical significance was achieved and mean values remained well within historical control data, the increase was considered not related to treatment.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment. All females had regular cycles of 4 days.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating index was considered not to be affected by treatment. The mating indices were 90% for the control and 100% for the 150, 375 and 750 mg/kg groups, respectively. Precoital time was considered not to be affected by treatment. Number of implantation sites was considered not to be affected by treatment. Fertility index was considered not to be affected by treatment. The fertility indices were 78%, 90%, 100% and 90% for the control, 150, 375 and 750 mg/kg groups, respectively.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

375 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment. For the pup of female no. 45 that was euthanized on Day 6, a pale appearance from Days 3-5 was noted and a lean appearance on Day 6. In addition, this pup had body weight of 4.7 grams, compared to a body weight of 8.0-9.1 grams in other pups of the same litter on PND 4. For the pup of female no. 48 that was euthanized on Day 11, an abnormal posture of the right foreleg was noted. The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 96%, 95%, 87% and 92% for the control, 150, 375 and 750 mg/kg groups, respectively. The survival index at 375 mg/kg was lower when compared with concurrent control, but remained within historical control data and in the absence of a dose response, this was considered unrelated to treatment. Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment. The live birth indices were 99%, 100%, 87% and 98% for the control, 150, 375 and 750 mg/kg groups, respectively. The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment. Litter size was considered not affected by treatment. Live litter sizes were 11.0, 11.1, 10.0 and 12.7 living fetuses/litter for the control, 150, 375 and 750 mg/kg groups, respectively. Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment. The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were considered not to be affected by treatment.
At 750 mg/kg, the body weights determined in seven available litters on PND 1-7 and six available litters at PND 13 remained similar to control values and historical control data. The data of this slightly lower number of litters was therefore considered adequate for evaluation.

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:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 750 mg/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.
For pup 6 of female no. 48 (control), a misshapen spinal cord was observed at macroscopy. For two pups of female no. 73 (750 mg/kg) that were found dead at first litter check, no milk in the stomach was noted. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.Gestation index and duration of gestation were considered not to be affected by treatment. The lactation indices were 96%, 98%, 100% and 100% for the control, 150, 375 and 750 mg/kg groups, respectively. Sex ratio was considered not to be affected by treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

development

Generation:

F1

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Aug 2018 - 14 Nov 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD guidance document supporting OECD test guideline 443 on the extended onegeneration reproductive toxicity test, No. 151

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals : 10 weeks

- Basis for dose level selection :
The dose levels in this study were selected to be 0, 10, 30, 100 mg/kg/day, based on the results of a preliminary reproductive toxicity study (combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD TG 422) with oral exposure of the test item in rats. In this preliminary study, animals were dosed with 0, 40, 125 and 375 mg/kg/day. From dose 125 mg/kg/day, adverse findings observed were ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females. Other test item-related findings consisted of an increase in liver weights in males and females and an increase in hyaline droplet accumulation in the male kidneys at 375 mg/kg/day. Based on the severe findings in the stomach, a parental local NOAEL was established of 40 mg/kg/day in this study. As animals are dosed for a longer time period for the Extended One-Generation Reproductive Toxicity Study (e.g. F0-males 11-13 weeks versus 4 weeks in an OECD 422 study), a maximum dose level of 100 mg/kg/day was selected.

- Inclusion/exclusion of extension of Cohort 1B : Inclusion

- Termination time for F2 : not applicable

- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B : Exclusion

- Inclusion/exclusion of developmental immunotoxicity Cohort 3 : Exclusion

- Route of administration : The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 6-7 week; (F1) 3 wks
- Weight at study initiation: (P) Males: 136 and 187 g; Females: 112 and 153 g; (F1) Males: mean 50 g; Females: 49 g
- Fasting period before study: During motor activity measurements, F0- female animals had no access to food for a maximum of 2 hours. During motor activity measurements, F0-female animals had no access to water for a maximum of 2 hours.
- Housing:
On arrival, prior to mating and during the post-weaning period, animals were group housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages (Macrolon type IV).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (type III)
During the post-mating phase, males were housed in their home cage (Macrolon type IV) with a maximum of 5 males/cage). Females were individually housed in Macrolon plastic cages (type III).
During the lactation phase, females were housed in Macrolon plastic cages (type III, height). Pups were housed with the dam until termination or weaning (on PND 21).
During locomotor activity monitoring, F0-females were housed individually in a Hi-temp polycarbonate cage
- Diet: ad libitum, Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum, Municipal tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 45 to 67
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared at least weekly as a solution, formulated in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 2, 6, 20 mg/mL
- Amount of vehicle: 5 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1:1
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages (type III).
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulations. The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Duration of treatment / exposure:

see table 1

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

25 (F0) / 20 (F1)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels in this study were selected based on the results of a preliminary reproductive toxicity study (combined 28-day repeated dose toxicity study with reproduction/developmental toxicity screening test according to OECD TG 422) with oral exposure of the test item in rats and in an attempt to produce graded responses to the test item. In this preliminary study, aninmals were dosed with 0, 40, 125 and 375 mg/kg bw/day. From dose 125 mg/kg bw/day, adverse findings observed were ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females. Other test item-related findings consisted of an increase in liver weights in males and females and an increase in hyaline droplet
accumulation in the male kidneys at 375 mg/kg/day. As animals are dosed for a longer time period for the Extended One-Generation Reproductive Toxicity Study (e.g. F0-males 11-13 weeks versus 4 weeks in an OECD 422 study), a maximum dose level of 100 mg/kg bw/day was selected. No (severe) clinical symptoms and effects on body weight and food consumption were noted (i.e. the general health of the animals was good). The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.

WATER CONSUMPTION AND COMPOUND INTAKE: No
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

FUNCTIONAL TESTS – F0-Generation
Functional tests were performed on the selected 5 females during Week 10 of treatment. The following tests were performed: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength, Locomotor activity

CLINICAL PATHOLOGY - F0-Generation and Cohort 1A animals
Sample collection:
Blood of 10 selected animals/sex/group of F0-animals and Cohort 1A animals was collected on the day of scheduled necropsy. Samples were collected from the retro-orbital sinus under anaesthesia using isoflurane in the animal facility.
The selected F0-animals and Cohort 1A animals were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available.
Urine was collected into a specimen vial from the 10 selected animals/sex/group of F0-animals and Cohort 1A animals housed in individual metabolism cages overnight (approximately 15-20 hrs) with absence of food, but water was available.

Oestrous cyclicity (parental animals):

Estrous stages were determined by examining the cytology of vaginal lavage samples. Daily vaginal lavage was performed for all F0-females beginning 14 days prior to mating and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of scheduled necropsy, a vaginal lavage was also taken.

Sperm parameters (parental animals):

Parameters examined in [F0, Cohort 1A] male parental generations:
Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. One epididymis (left) was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing, the left epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.

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); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:

F1-Generation until Weaning (PND 21):

Mortality/Moribundity Checks
Pups were observed twice daily for general health/mortality, simultaneously with the mortality/moribundity check of the dam. The number of live and dead pups was determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations
Clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check were given in the respective report tables.

Body Weights
Live pups were weighed individually on PND 1, 4, 7, 13 and 21. For animals of Cohort Surplus, a terminal weight was recorded on the day of scheduled necropsy.

Sex
Sex was externally determined for all pups on PND 1 and 4 (sex determination not noted for Day 13)

Anogenital Distance
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.

Areola/Nipple Retention
All male pups in each litter were examined for the number of areola/nipples on PND 13.

Clinical Pathology
On PND 4 at culling, blood was collected from two surplus pups per litter (from all litters, if possible) by decapitation for determination of thyroid hormone.

F1-Generation from Weaning (PND 21) onwards:

Mortality/Moribundity Checks – Cohorts 1A, 1B and 1C
Throughout the study, animals were observed for general health/mortality and moribundity twice daily. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations– Cohorts 1A, 1B and 1C
Clinical observations were performed at least twice daily, up to the day prior to necropsy. These observations were at least conducted prior to dosing and 0-30 minutes after dosing.

Body Weights – Cohorts 1A, 1B and 1C
Animals were weekly weighed individually. This started on a specific date on which all pups were at least at PND 21. In addition, the body weight was recorded of each female on the day of acquisition of vaginal patency and of each male on the day of acquisition of balanopreputial separation. For animals of Cohorts 1A and 1B, a terminal weight was recorded on the day of scheduled necropsy.

Food Consumption– Cohorts 1A, 1B and 1C
Food consumption was quantitatively measured weekly, from weaning onwards up to the day prior to scheduled necropsy.

Water Consumption – Cohorts 1A, 1B and 1C
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

Vaginal Patency – Cohorts 1A, 1B and 1C
Vaginal patency (vaginal opening) was monitored daily for all females from PND 25 onwards until vaginal patency was present, by visual inspection of the vaginal area.

Balanopreputial Separation – Cohorts 1A, 1B and 1C
Balanopreputial separation (prepuce opening) was monitored daily for all males from PND 35 onwards until balanopreputial separation was present, by visual inspection of the genital area.

Stage of Estrus Determination – Cohorts 1A, 1B and 1C
Estrous stages were determined by examining the cytology of vaginal lavage sample, taken on the day of scheduled necropsy.

Estrous Cycle Determination – Cohort 1A
Estrous stages were determined by examining the cytology of vaginal lavage samples, taken during two periods. During the first period, daily vaginal lavage was performed for all Cohort 1A females starting on the day of onset of vaginal patency and was minimally continued until the first estrus was determined, in order to determine the time interval between these two events. During the second period, daily vaginal lavage was performed from PND 75 to 88.

Clinical Pathology - F1- animals of Cohort Surplus on PND 22
On PND 22, blood was collected from all Cohort Surplus animals (10/sex/group), if possible for determination of Thyroid Hormone. Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure.

Clinical Pathology – Cohort 1A
Blood of 10 selected animals/sex/group of F0-animals and Cohort 1A animals was collected on the day of scheduled necropsy for
- Hematology: White blood cells (WBC), Red Blood Cell Distribution Width (RDW), Neutrophils (absolute), Haemoglobin, Lymphocytes (absolute), Haematocrit, Monocytes (absolute), Mean corpuscular volume (MCV), Eosinophils (absolute), Mean corpuscular haemoglobin (MCH), Basophils (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells, Platelets, Reticulocytes (absolute), Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)
- Clinical Chemistry: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT),Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Bile Acids, Calcium, Urea, Inorganic Phosphate (Inorg. Phos)
- Thyroid hormone: Thyroxine (T4), Thyroid Stimulating Hormone (TSH)
Urinalysis: Specific gravity, White blood cells (WBC-sed.), Clarity Red blood cells (RBC-sed.), Colour Casts, pH, Epithelial cells, Blood Crystals, White blood cells (WBC), Bacteria, Bilirubin, Urobilinogen, Protein, Ketones, Glucose, Nitrite

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

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: yes
From 10 selected animals/sex/group of Cohort 1A, splenic lymphocyte subpopulation analysis was performed at termination. If possible, one pup (male or female) was selected per litter (20 litters in total). One half of the spleen was kept on ice until splenic lymphocytes were isolated using 70 μm cell strainers. The other half of the spleen was preserved for histopathological evaluation. Splenocytes were counted with the Coulter Counter Z1. The following subpopulations were determined in isolated splenic lymphocytes using the BD FACSCanto™ flow cytometer system on the day of necropsy:
T-cells, T-helper cells, T-cytotoxic cells, B-cells, NK-cells, Ratio T-helper cells/ T-cytotoxic cells (Th/Tc)
The % lymphoid cells of peripheral blood mononuclear cells (PBMC) were determined using the Forward Scatter and Side Scatter.

Postmortem examinations (parental animals):

SACRIFICE- F0 Generation
Animals surviving until scheduled euthanasia were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
Scheduled necropsies:
Males (which sired and failed to sire): After successful mating and a minimum of 10 weeks of treatment.
Females which delivered: LD 23-25.
Females which failed to deliver:
With evidence of mating: Post-coitum Days 25-27 (102,105, 108, 111, 122, 144, 179, 194, 200).
Without evidence of mating: 24 days after the last day of the mating period (Nos. 112, 168)
Females with total litter loss (No. 126): Within 24 hours after the last pup was found dead or missing.

Except for females with total litter loss, all animals surviving to scheduled necropsy were fasted overnight with a maximum of approximately 24 hours before necropsy. Water was available.

GROSS NECROPSY- F0 Generation
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females. In case no
macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

HISTOPATHOLOGY / ORGAN WEIGHTS - F0 Generation
The organs identified in Table 5 were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios were calculated. Representative samples of the tissues identified in Table 5 were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution), unless otherwise indicated.

Postmortem examinations (offspring):

SACRIFICE -F1 Generation until weaning
Unscheduled Deaths– F1-Generation
Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally, if possible) and externally examined with emphasis on developmental morphology. For pups found dead from PND 14 onwards a limited necropsy was performed including sex determination (both externally and internally, if possible). Descriptions of all external abnormalities were recorded. External abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director. The
stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Culled Pups (PND 4) – F1-Generation
On PND 4, the pups scheduled for culling (> 8 pups per litter) were euthanized by decapitation.

GROSS NECROPSY
See table 3 for details

Cohort 1A
Scheduled necropsy of Cohort 1A was conducted on PND 89-95. All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

Cohort 1B
Scheduled necropsy of Cohort 1B was conducted on ≥ PND 97. Cohort 1B animals were not deprived of food overnight before necropsy. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

Cohort 1C
Scheduled necropsy of Cohort 1C was conducted after positive determination of vaginal patency or balanopreputial separation. Cohort 1C animals were not deprived of food overnight before necropsy and no terminal body weight was recorded. All
animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

Cohort Surplus
Scheduled necropsy of Cohort Surplus was conducted on PND 22. Cohort Surplus animals were not deprived of food overnight before necropsy and a terminal body weight was recorded. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGTHS
Cohort 1A
In addition to the procedures described above, HE stained step sections of ovaries and corpora lutea at a thickness of 5 micrometers (5 step sections in total, including the routine section) were prepared for the Cohort 1A animals of Group 1 and 4 for quantitative evaluation of follicles (primordial and small growing follicles counted together), as well as corpora lutea.
Cohort 1B
In addition to the procedures described above, the reproductive organs of all Cohort 1B animals were processed to block stage.

Statistics:

Parametric:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric:
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). The motor activity data set (at least 3 groups) was compared using an overall Kruskal-Wallis.
Incidence:
An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

see table 4

Offspring viability indices:

see table 4

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test-item related clinical signs were noted up to treatment with 30 mg/kg bw/day. No findings were noted during the weekly arena observations in this study.
Salivation seen after dosing among animals of the 100 mg/kg bw/day dose group during the first 8 weeks of the treatment period was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than being a sign of systemic toxicity. Other clinical signs noted during the treatment period occurred within the range of
background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

No test item-related mortality occurred during the study period. One female of the 10 mg/kg bw/day group was euthanized on Lactation Day 1, as she had a total litter loss.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gain were considered to have been unaffected by treatment up to 100 mg/kg bw/day. A trend towards decreased body weights and body weight gain may appeared in males at 100 mg/kg bw/day up to Week 1 of the mating period. A statistical significant decrease in bodyweight was observed at 100 mg/kg bw/day in Week 8 of treatment. The statistical significant increase in body weight gain at 30 mg/kg bw/day in Week 3 of mating was considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption before or after correction for body weight in animals treated up to 100 mg/kg bw/day was similar to the control level over the treatment period. Any statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment.

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):

No toxicologically relevant changes were noted in haematological parameters. Statistically significant lower white blood cell count (WBC) was noted in females at 10 and 100 mg/kg bw/day, which was likely the result of lower lymphocytes levels observed in these animals. In absence of a dose response and as values remained within normal range, these changes were considered unrelated to treatment with the test item. Any other statistically significant changes in haematology parameters achieving a level of statistical significance when compared to controls were considered unrelated to administration of the test item due to the minimal magnitude of the change and/or absence of a dose response. Coagulation parameters of treated rats were considered not to have been affected by treatment up to 100 mg/kg bw/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes were observed in treated males at 30 and/or 100 mg/kg bw/day:
Increase in urea at 100 mg/kg/day (1.16x, 4.3 mmol/L).
• Increase in sodium at 30 and 100 mg/kg bw/day (1.01x (143.3 mmol/L) and 1.02x (143.8 mmol/L), respectively).
• Increase in potassium at 100 mg/kg bw/day (1.07x, 4.02 mmol/L)
Any other statistically significant changes in clinical chemistry parameters achieving a level of statistical significance when compared to controls, occurred in the absence of a dose-related response. As such, these slight differences were considered not related to treatment with the test item. Serum levels of TSH and T4 in F0-males and -females were not affected by treatment up to 100 mg/kg bw/kg.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis was not affected by treatment with the test item up to 100 mg/kg bw/day.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals. Motor activity (total movements and ambulations) was (non-statistically) slightly increased in high dose animals. This slight increase was considered due to one female, for which a level of total movements and ambulations was recorded.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic alterations. All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. Histopathological examination of reproductive tissues revealed no evidence of a treatment-related effect on reproduction.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were unaffected by treatment with the test item. All females had regular cycles of 4 to 5 days.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Sperm motility, concentration and morphology parameters were unaffected by treatment up to 100 mg/kg bw/day. Statistically significant changes noted were considered unrelated to treatment as a dose-related response was absent, and since the opposite effect would be expected in case of toxicity.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating index was considered not to be affected by treatment. With the exception of one control and one 30 mg/kg bw/day female, all females showed evidence of mating. Precoital time was considered not to be affected by treatment. With the exception of one female (mated at Day 14), all females showed evidence of mating within the first 4 days. Number of implantation sites was considered not to be affected by treatment up to 100 mg/kg bw/day. Fertility index was considered not to be affected by treatment. The fertility indices were 79%, 96%, 100% and 88% for the control, 10, 30 and 100 mg/kg bw/day groups, respectively. A total of 5 control females, one female at 10 mg/kg bw/day and 3 females at 100 mg/kg bw/day were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was considered not to be related to treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Remarks on result:

other: A dose level of 100 mg/kg bw/day was selected as the NOAEL of 40 mg/kg bw/day was established in a dose range finder. Here, adverse findings consisted of ulcers and inflammation of the forestomach and hyperplasia squamous cells from 125 mg/kg bw/day on.

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest doses tesed

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment. For the pup of one Female (10 mg/kg bw/day) which was missing on Day 2, absence of milk in the stomach was noted on Day 1 and for the pup of Female No. 154 which was missing on Day 2, a pale appearance was noted on Day 1. The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant. No test item-related clinical signs were noted during daily detailed clinical observations in Cohort 1A,1B and 1C or during weekly arena observations.

Mortality / viability:

no mortality observed

Description (incidence and severity):

No mortality occurred during the study period in Cohort 1A, 1B and 1C from weaning onwards that was considered to be related to treatment with the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights of pups were considered not to be affected by treatment.
Body weights and body weight gains of Cohort 1A, 1B and 1C were considered unaffected by treatment up to 100 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before and after correction for body weight was considered unaffected by treatment up to 100 mg/kg bw/day.
Any statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment as changes were only minimal and/or no trend was apparent regarding dose and duration of treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A: The following statistically significant changes were noted in male animals at 100 mg/kg bw/day.
• Increased neutrophil levels (1.3x)
• Increased lymphocyte levels (1.16x)
The statistically significant change in white blood cell count (WBC) in males at 10 mg/kg bw/day was considered not to be related to treatment with the test item as it occurred in the absence of a dose-related trend. Coagulation parameters of treated rats were considered not affected by treatment up to 100 mg/kg bw/day.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female pups, culled at PND 4 and male and female pups of Cohort Surplus at PND 22 were considered not to be affected by treatment. Serum TSH levels in male and female pups of Cohort Surplus at PND 22 were considered not to be affected by treatment.
Cohort 1A: Clinical chemistry parameters of treated rats were considered not affected by treatment up to 100 mg/kg bw/day. Serum levels of thyroid stimulating hormone (TSH) and Total T4 were considered not to be affected by treatment up to 100 mg/kg bw/day.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Cohort 1A: Urinary parameters were unaffected by treatment.

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

Balanopreputial separation and vaginal patency were considered unaffected by treatment up to 100 mg/kg bw/day. At 30 mg/kg bw/day, an increase in the age of reaching balanopreputial separation was observed (1.03x compared with control). This was likely related to Male No. 355 that had a very low body weight and had reached balanopreputial separation at age PND 54 (versus average age of reaching balanopreputial separation on PND 41.5 for control animals).
At 10 mg/kg/day, an increase in age of reaching first estrus (1.04x compared with controls) was noted in females. In absence of a dose response, this slight increase was considered unrelated to treatment.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 100 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related organ weight changes in the Cohort surplus-animals.

Cohort 1A and 1B:There were no test item-related organ weight changes observed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups sacrificed at the end of the lactation period that were considered to be related to treatment.
Cohort 1A and 1B: There were no test item-related macroscopic alterations in the F1-generation. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. Watery fluid in the uterus, found in several females in all dose groups in Cohort 1A and 1B, is related to a stage in the estrous cycle and is a normal finding.

Histopathological findings:

no effects observed

Description (incidence and severity):

Cohort 1A: There were no test item-related microscopic alterations in Cohort 1A of the F1-generation.. All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Spermatogenesis-staging (Cohort 1A)
Stage-dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Ovarian Follicle Counts - Cohort 1A
There were no test item-related effects on the ovarian follicle counts in the F1-females (Cohort 1A) at 100 mg/kg/day when compared to control group females. Any variation between group mean counts represented biological variability and were not statistically significant.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Estrous Cycle – F1-Generation (Cohort 1A):
Length and regularity of the estrous cycle were considered not to have been affected by treatment. Most females had regular cycles of 4 to 5 days. Female No. 636 (30 mg/kg bw/day) had two cycles with a duration of 2 days and one of 5 days and was therefore classified as having an irregular cycle. For Female Nos. 495 (control) and 555 (10 mg/kg bw/day) estrous cycle regularity/length could not be determined. Considering the low incidence and in absence of a dose response, this finding was considered unrelated to treatment with the test item.
Sperm Analysis – F1-Generation (Cohort 1A):
At 100 mg/kg bw/day, an increase in spermcount of the epididymides was observed of 1.23x compared with controls. This was considered unrelated to treatment as a dose-related response was absent, and since the opposite effect would be expected in case of toxicity.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

Cohort 1A: There were no test item-related effects on splenic lymphocyte subpopulations observed up to 100 mg/kg bw/day.
Higher T-cell and T-cytotoxic cell splenic subpopulations and lower B-cell splenic subpopulations observed for females reached statistical significance when compared with controls at 10 mg/kg bw/day. However, these shifts were slight of nature and occurred in the absence of a dose-related response and in absence of any test item-related microscopic findings in the spleen. As such, these shifts were considered to represent biological variability and were considered not to be related to treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Reference 3

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For Read Across Justification please refer to Section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Remarks on result:

other: A dose level of 100 mg/kg bw/day was selected as the NOAEL of 40 mg/kg bw/day was established in a dose range finder. Here, adverse findings consisted of ulcers and inflammation of the forestomach and hyperplasia squamous cells from 125 mg/kg bw/day on.

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Reference 4

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

01 Mar 2018 - 16 July 2018

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:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: males were 10 weeks old, females were 13 weeks old
- Weight at study initiation: males weighed between 251 and 322 g, females weighed between 198 and 263 g
- Housing:
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type). Pups were housed with the dam.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage enrichment, bedding material, food and water.
- Diet: ad libitum, Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum, Municipal tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22
- Humidity (%): 36 to 60
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was selected as vehicle, at request of the Sponsor and based on previously performed studies.
- Concentration in vehicle: 5 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- Once mating had occurred, the males and females were separated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Duration of treatment / exposure:

Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-62 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 40-53 days.

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

375 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 16-day dose range finder with oral gavage administration of the test item in rats and in an attempt to produce graded responses to the test item.

- Fasting period before blood sampling for clinical biochemistry: yes

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION: Yes
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION AND COMPOUND INTAKE: No data
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was expected or noted at visual inspection of the water bottles.

FUNCTIONAL TESTS
Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 9-12). These tests were performed after dosing, after completion of clinical observations and arena observations, if applicable. The following tests were performed: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength, Locomotor activity

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.

Sperm parameters (parental animals):

Parameters examined in all male parental generations:
testis weight, epididymis weight, spermatogenic profiling

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); Blood samples were collected from two of the surplus pups (if possible from one male and one female pup).

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:

Clinical Observations: Clinical observations were performed at least once daily for all pups. Pups were observed daily for general health/mortality. The number of live and dead pups was determined on PND 1 and daily thereafter.
Body Weights: Live pups were weighed individually on PND 1, 4, 7 and 13.
Anogenital Distance: Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
Areola/Nipple Retention: All male pups in each litter were examined for the number of areola/nipples on PND 13.

GROSS EXAMINATION OF DEAD PUPS:
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally). Pups found dead during the weekend were fixed in identified containers containing 70% ethanol as they were not necropsied on the same day. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
Scheduled necropsies were conducted on the following days:
Males (which sired and failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
Females which delivered: PND 14-16.
Females which failed to deliver: With evidence of mating: Post-coitum Day 25 or 26
Without evidence of mating: 25 days after the last day of the mating period
Females with total litter loss: Dams with no surviving pups were euthanized within 24 hours after the last pup was found dead or missing.

All males surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0-females were not fasted.

GROSS NECROPSY
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
see table 1 and 2

The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
Selected animals: Tissues identified in Table 2 (except animal identification, aorta, nasopharynx, esophagus, harderian gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue).
Males that failed to sire (except for males which were selected), females that failed to deliver pups and females with total litter loss: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina
Females with total litter loss: Mammary gland
Remaining animals: Gross lesions/masses

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day of necropsy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: only males
- How many animals: 5/sex/group
- Parameters checked: White blood cells (WBC), Neutrophil (absolute), Lymphocyte (absolute), Monocyte (absolute), Eosinophil (absolute), Basophil (absolute), Red blood cells, Reticulocyte (absolute), Red Blood Cell Distribution Width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet, Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)

For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no adverse changes in T4 were noted in F0-males, no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of necropsy
- Animals fasted: only males
- How many animals: 5/sex/group
- Parameters checked: Alanine aminotransferase (ALAT), Glucose, Aspartate aminotransferase (ASAT), Cholesterol, Alkaline Phosphatase (ALP), Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Urea, Calcium, Creatinine, Inorganic Phosphate (Inorg. Phos)

Postmortem examinations (offspring):

SACRIFICE
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

GROSS NECROPSY
Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.

On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by decapitation, between 7.00 and 10.30 a.m., and samples were pooled to one sample per litter. If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much blood as possible was collected from this single pup. On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female). Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16.

Statistics:

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

see table 5

Offspring viability indices:

see table 5

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations. Salivation seen after dosing among animals of all dose groups was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

No treatment-related mortalities occurred. One female of the control group was euthanized on Lactation Day 1, as she had a total litter loss (at first litter check she had only dead pups).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gain were considered to have been unaffected by treatment. Body weight gain was increased in males in the second week of treatment after dosing of 125 and 375 mg/kg, and in the fifth week of treatment after dosing with 375 mg/kg. These changes in body weight gain were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment and values were within the historical control range.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after correction for body weight were recorded. The statistically significant change in relative food consumption in 40 mg/kg females during post-coitum Days 17-20 was considered to be unrelated to treatment, since no trend was apparent regarding dose and duration of treatment.

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):

No toxicologically relevant changes were noted in haematological parameters. A decreased number of reticulocytes was observed in females at 125 mg/kg bw/day, which was considered unrelated to administration of the test item due to absence of a dose-related trend response. Coagulation parameters (prothrombin time and activated partial thromboplastin time) of treated rats were considered not to have been affected by treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant increases were noted for treated males (relative changes in mean values as compared to the concurrent control group are indicated between parentheses):
-increased total protein at 375 mg/kg (6%)
-increased albumin at 375 mg/kg (6%)
-increased calcium at 375 mg/kg (4%)
-increased urea at 40 and 375 mg/kg (27% and 30%, respectively)
The changes in total protein, albumin and calcium were minimal and remained within the historical control range. No dose related trend was observed for the increase in urea and values remained within the historical control range. In addition, a slight increase in sodium was observed at 125 mg/kg (1%), which occurred in the absence of a dose related trend. No treatment-related changes were noted in clinical chemistry parameters in females.

Thyroid hormone analyses:
Serum levels of T4 in F0 males were increased at 125 and 375 mg/kg (35% and 37% increase in mean values compared to concurrent control, respectively). These values remained within the historical control range and the control value was on the lower limit of this range. In addition, no effect was observed in respect to thyroid weight, therefore, this effect was considered not toxicologically relevant.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation parameters were not considered to be affected by treatment in males up to 375 mg/kg bw/day.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Forelimb and hind limb grip strength was similar between control and treated animals. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment were noted in the (fore)stomach of males and females and the liver and kidneys of males:
Stomach, squamous cell hyperplasia: was present in 2/5 males starting at 125 mg/kg bw/day up to marked degree and in females at 375 mg/kg bw/day up to moderate degree. This correlated with the macroscopic irregular surface.
Stomach, ulcer forestomach: was present in males starting at 125 mg/kg bw/day at minimal degree.
Stomach, inflammation forestomach: was present in males starting at 125 mg/kg bw/day up to moderate degree.
Liver, hepatocellular hypertrophy: was present in males treated at 375 mg/kg bw/day at minimal degree. This correlated with the increased liver weight.
Kidneys: an increased incidence and severity of hyaline droplet accumulation was present in males treated at 375 mg/kg bw/day up to slight degree.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were not considered to have been affected by treatment.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating index was not considered to be affected by treatment. The mating indices were 90%, 100%, 90% and 100% for the control, 40, 125 and 375 mg/kg groups, respectively. Precoital time was not considered to be affected by treatment. The mean precoital times were 2.6, 2.9, 2.6 and 4.5 for the control, 40, 125 and 375 mg/kg groups, respectively. The (non-statistically) higher mean precoital time for group 375 mg/kg was considered unrelated to treatment as 8 females were mated within the first three days of mating and the median precoital time was 3 for all dose groups. Number of implantation sites was not considered to be affected by treatment. Fertility index was not considered to be affected by treatment.
The fertility indices were 100%, 90%, 78% and 90% for the control, 40, 125 and 375 mg/kg groups, respectively. A total of 4 females were not pregnant (one at dose 40 and 375 mg/kg and two females at dose 125 mg/kg). Since these cases of non-pregnancy showed no dose-related incidence across the dose groups, as the incidences were within normal limits, and given the absence of any reproductive/developmental toxicity, this was not considered to be related to treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

375 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

375 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.
For the pup of one female in group 375 mg/kg bw/day who was missing on Day 4, less milk in the stomach and a lean appearance were noted on Day 1. Alopecia of the whole body was noted for several pups of the control, 40 and 375 mg/kg groups. The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment. Viability indices were 99%, 100%, 99% and 99% for the control, 40, 125 and 375 mg/kg groups, respectively. One pup of the control group (litter 48) was killed in extremis on Day 1 of lactation. One pup at 125 mg/kg and one pup at 375 mg/kg were missing on PND 4. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. Litter size was not considered affected by treatment.
Live litter sizes were 10.9, 9.2, 12.1 and 11.6 living pups/litter for the control, 40, 125 and 375 mg/kg groups, respectively.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were not considered to be affected by treatment.

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:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were not considered to be affected by treatment

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 375 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not considered to be affected by treatment. No pups were found dead/missing between lactation Days 5 and 13, resulting in a lactation index of 100% for all groups. Gestation index and duration of gestation were not considered to be affected by treatment. Except for one female (no. 49) of the control group, all pregnant females had live offspring. The gestation indices were 89% for the control and 100% for the 40, 125 and 375 mg/kg groups, respectively. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed. The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 92%, 79%, 88% and 85% for the control, 40, 125 and 375 mg/kg groups, respectively. The slightly lower post-implantation survival index for the 40 mg/kg group was considered the result of the low litter size of female no. 51 which only had one pup, but 10 implantation sites. The survival indices for all other groups were considered within normal range.Sex ratio was not considered to be affected by treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F1

Effect level:

375 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Reference 5

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

For Read Across Justification please refer to Section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

375 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

375 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F1

Effect level:

375 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

OECD 443 (Read-Across)

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The substance was tested in a Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD TG 422 and in compliance with GLP (ReachCentrum, 2019A). Wistar Han rats were treated by daily oral gavage at dose levels of 150, 375 and 750 mg/kg bw/day. The rats of the control group received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 14-16 days of lactation (for 50-58 days). Females that failed to deliver pups were treated for 39-54 days. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional tests, body weight and food consumption, estrous cycle length and regularity, clinical pathology, serum level of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examination. In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio, live litter size and early postnatal pup development (mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, serum level of thyroid hormone T4 in PND 14-16 pups and macroscopy). Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

Parental results:

Microscopic evaluation showed local alterations of the forestomach starting at 150 mg/kg in males (non-adverse at this dose level) and at 375 mg/kg in females (non-adverse at this dose level), which consisted of ulcers and/or lymphogranulocytic inflammation and/or squamous cell hyperplasia and/or hyperkeratosis and/or edema in the submucosa. In addition, ulcers which were degenerative in nature were noted in males at 375 mg/kg and at 750 mg/kg (in both males and females). In a dose-related incidence, an irregular surface of the stomach was noted at necropsy in treated animals. In addition, other corroborative findings included salivation observed in treated animals and increased neutrophil values noted in males at 750 mg/kg. The local forestomach effects were considered to be adverse at 375 mg/kg (males) and 750 mg/kg (both sexes). At 750 mg/kg, two males and two females were sacrificed in extremis and one female was found dead. For one male and one female, test item-related findings in the forestomach were considered to be the main cause of moribundity. For the other male the cause of mortality could not be established. For the two remaining females the observed mortality was considered to be related to the oral gavage procedure. At 375 mg/kg, the death of a single female was to be considered incidental. Other changes noted in treated rats, mostly at 750 mg/kg, were considered non-adverse as discussed below. In males, a treatment-related reduction in body weight gain was noted at 750 mg/kg, corresponding to a decreased food consumption during the first week of treatment. As the reductions in mean body weight (gain) and food consumption remained slight and appeared to recover in subsequent intervals, the differences were considered to be not adverse. Test item related increases in mean kidney and liver weights were observed at 375 (kidneys) and at 750 mg/kg (kidneys and liver). The increase in liver weights was correlated with centrilobular hepatocellular hypertrophy observed at microscopic examination. In absence of any degenerative or inflammatory changes, the slight change in liver weights was considered non-adverse. For the increase in kidney weight, no microscopic correlate was found and was considered to be non-adverse. An increased incidence and severity of extramedullary hematopoiesis was observed in females at 750 mg/kg. At the incidence and severity observed, this was considered to be a non-adverse finding. Exposure to the test item was not associated with obvious toxicity in the remaining parental parameters examined in this study (i.e. functional observations and clinical laboratory investigations (including male T4 thyroid hormone levels)).

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (750 mg/kg bw/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Developmental results:

In the control group, seven instead of the intended eight litters were available for evaluation. Sufficient information was available from control litters and historical control data for a thorough toxicological evaluation. No developmental toxicity was observed up to the highest dose level tested (750 mg/kg). No toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following no-observed-adverse effect level (NOAEL) of the substance were established:

Parental (systemic) NOAEL: 375 mg/kg bw/day, based on treatment related mortality at 750 mg/kg bw/day.

Parental (local) NOAEL: 150 mg/kg bw/day, based on adverse local forestomach effects at 375 mg/kg bw/day (males) and 750 mg/kg bw/day(both sexes).

Reproduction NOAEL: at least 750 mg/kg bw/day.

Developmental NOAEL: at least 750 mg/kg bw/day.

 

Furthermore, a study according OECD TG 443 with the read-across substance (CAS 42978-66-5, ReachCentrum, 2019C) is available which was conducted to provide an evaluation of possible pre- and postnatal effects on development. In addition, a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats was performed

Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. In addition, the study provided and/or confirmed information about the effects of the substance on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behaviour, conception, pregnancy, parturition, and lactation.

This Extended One Generation Reproductive Toxicity Study included Cohort 1 for assessment of reproductive/developmental toxicity. Wistar Han rats were treated with the test item by daily oral gavage at dose levels of 10, 30 and 100 mg/kg bw/day. The animals of the control group received the vehicle, Corn Oil, alone. F0-males were treated for 11-13 weeks, including 10 weeks prior to mating and during the mating and post-mating period, up to and including the day before scheduled necropsy. F0-females were treated for a minimum of 14-18 weeks, including 10 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least 21 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 14 weeks. From weaning onwards (PND 21), F1-animals of Cohorts 1A, 1B and 1C, were dosed up to and including the day before scheduled necropsy (i.e. for a minimum of 3 weeks). The F1-animals of Cohort Surplus and Spares (not assigned to one of the cohorts) were not dosed.

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

F0-Generation:

No parental toxicity was observed up to the highest dose level tested (100 mg/kg bw/day). At 30 and 100 mg/kg bw/day, a minor increase in sodium was noted in males and additionally, at 100 mg/kg bw/day, urea and potassium levels were slightly increased. As values were only slightly above the range considered normal (sodium) or remained within normal range (urea and potassium) and no corroborative microscopic findings were found, these changes were considered non-adverse. No test item-related changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations (females), body weight, food consumption, haematology, coagulation, thyroid hormone analysis, urinalysis, macroscopic examination, organ weights and microscopic examination).

Reproductive results – F0-generation

No reproduction toxicity was observed up to the highest dose level tested (100 mg/kg bw/day). No test item-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, sperm analysis, and histopathological examination of reproductive organs including stage-dependent qualitative evaluation of spermatogenesis in the testis).

Developmental results – F0-generation / F1-generation (pre-weaning)

No developmental toxicity was observed up to the highest dose level tested (100 mg/kg/day) during the pre-weaning phase. No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, duration of gestation, viability and weaning indices, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, thyroid hormone levels (T4 of PND 4 and 22 pups and TSH of PND 22 pups), and macroscopic examination).

Developmental results – F1 generation (post-weaning)

No developmental toxicity was observed up to the highest dose level tested (100 mg/kg bw/day) during the post-weaning phase. At 100 mg/kg bw/day, increases in neutrophil and lymphocyte levels were found in males. As values remained within normal range and no correlative microscopic findings were found, these changes were considered non-adverse. No test item-related changes were noted in any of the other developmental parameters investigated in this study (i.e. mortality, clinical signs, body weight, food consumption, coagulation, clinical chemistry, thyroid hormone analysis, urinalysis, splenic lymphocyte subpopulation, balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first estrous, time between vaginal opening and first estrous length and regularity of the estrous cycle, sperm analysis, macroscopic examination, organ weights, ovarian follicle and corpora lutea counts, and microscopic examination, including stage-dependent qualitative evaluation of spermatogenesis in the testis).

Higher doses could not be tested as these were not tolerated (according to the Dose-Range Finder Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test). In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1), the following no-observed-adverse-effect levels (NOAEL) of the read across substance were established:

General toxicity (F0 and F1): at least 100 mg/kg bw/day

Reproduction: at least 100 mg/kg bw/day

Developmental: at least 100 mg/kg bw/day

A maximum dose level of 100 mg/kg bw/day was selected as a NOAEL of 40 mg/kg bw/day was established in a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test with oral exposure of the read across substance in rats. In this preliminary study, adverse findings consisted of ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females from 125 mg/kg bw/day.

In order to support the read-across approach for reproductive toxicity additional reproductive toxicity screening assays are available:

 

A study according to OECD TG 422 was conducted to determine the potential toxic effects of the structural analogue substance (CAS 42978-66-5, ReachCentrum, 2019B) when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated. Wistar Han rats were treated with the substance by daily oral gavage at dose levels of 40, 125 and 375 mg/kg bw/day. The rats of the control group received the vehicle, Corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-62 days). Females that failed to deliver pups were treated for 40-53 days. Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

Parental results:

Parental toxicity was observed in the (fore)stomach of males from 125 mg/kg and in females at 375 mg/kg. These changes consisted of ulcers and inflammation of the forestomach in males and

hyperplasia squamous cells in males and females. Other treatment-related but non-adverse changes were observed in the liver at microscopic examination. An absolute increase of 31% and a relative increase of 25% in liver weight was observed at dose 375 mg/kg. At microscopic examination, hepatocellular hypertrophy in the liver was observed at minimal severity and was in the absence of any indicators of cellular degeneration. The changes in the liver were not considered adverse at current severities. In the kidneys an increase in hyaline droplet accumulation was recorded in males which was considered to likely represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules (Alden et al., 1991). This male rat specific protein is not present in female rats nor in higher mammals, including man (Sahota et al., 2013). The increased hyaline droplet accumulation in the male kidneys at 375 mg/kg/day was not accompanied by indicators of tubular damage and therefore this was considered to be non-adverse. Functional observations were not performed for females and therefore, possible treatment related effects on the functional parameters could not be evaluated. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations (males), body weight, food consumption, clinical laboratory investigations (including male T4 thyroid hormone levels), macroscopic examination and organ weights).

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (375 mg/kg bw/day).

Developmental results:

No developmental toxicity was observed up to the highest dose level tested (375 mg/kg bw/day).

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the NOAEL of the read-across substance were established:

Parental local NOAEL: 40 mg/kg (based on findings in the (fore)stomach)

Parental systemic NOAEL: at least 375 mg/kg bw/day

Reproduction NOAEL: at least 375 mg/kg bw/day

Developmental NOAEL: at least 375 mg/kg bw/day

 

A study according to OECD TG 422 was also performed with the structural analogue substance Propylidynetrimethanol, ethoxylated, esters with acrylic acid (CAS 28961 -43 -5, ReachCentrum, 2019), when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. The dose levels in this study were selected to be 0, 100, 300 and 1000 mg/kg/day.

Parental results:

Microscopic evaluation showed local alterations of the forestomach starting at 100 mg/kg/day in males and at 300 mg/kg/day in females, which consisted of diffuse squamous cell hyperplasia and a lymphogranulocytic inflammation or a focal ulceration. At 300 mg/kg/day (males) and 1000 mg/kg/day (both sexes), these findings were accompanied by prominent hyperkeratosis, (sub) mucosal edema (up to moderate in males and slight in females) and erosions (both sexes up to slight). In mid and high-dose animals, an irregular surface of the stomach was noted at necropsy. In addition, corresponding clinical symptoms included salivation (at 300 and 1000 mg/kg/day) and rales (at 1000 mg/kg/day only). The local forestomach effects were considered to be adverse at 300 mg/kg/day (males) and 1000 mg/kg/day (both sexes).

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (375 mg/kg bw/day).

Developmental results:

No developmental toxicity was observed up to the highest dose level tested (375 mg/kg bw/day). In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAEL) for Propylidynetrimethanol, ethoxylated, esters with acrylic acid were established:

Parental (systemic) NOAEL: 1000 mg/kg/day

Parental (local) NOAEL: 100 mg/kg/day, based on adverse local forestomach effects at

300 mg/kg/day (males) and 1000 mg/kg/day (both sexes).

Reproduction NOAEL: 1000 mg/kg/day

Developmental NOAEL: 1000 mg/kg/day

Conclusion

Based on the effects observed in the combined repeated dose toxicity studies available for the target and the source substances it is concluded that the target substance is not expected to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. In all studies, no impairment of fertility and development of the offspring were observed up to the highest dose tested and tolerability of the substances was limited by local effects in the (fore)stomach rather than any systemic signs of toxicity.

Effects on developmental toxicity

Description of key information

A study equivalent or similar to OECD TG 414 and designed to screen potential maternal, embryotoxic and teratogenic effects of the test item in the rat was performed (Cytec, 1984). The test item produced observable maternal toxicity at a dose level of 1000 mg/kg bw/day but did not induce a teratogenic or embryotoxic effect in rats.

A prenatal developmental toxicity study according to OECD TG 414 on rabbits is available for the structural analogue CAS 42978-66-5 (ReachCentrum, 2018). Since adverse effects were neither observed for maternal nor for developmental toxicity, a maternal and developmental No Observed Adverse Effect Level (NOAEL) of at least 450 mg/kg was established.

A further study on developmental toxicity in rats with the structural analogue substance CAS 28961-43-5 is available. In conclusion, adverse effects on the developmental offspring were neither seen in rats nor in rabbits up to the highest dose tested.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June, 28, 1984 to December 11, 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

other: Sprague Dawley COBS CD rats

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding laboratories, Inc., Portage, Michigan
- Age at study initiation: 13 wk
- Weight at study initiation: 225- 297 g (on Day 0 of gestation)
- Housing: Individually housed in wire mesh cages suspended above cage board
- Diet: Purina certified rodent chow; ad libitum
- Water: Tap water; ad libitum
- Acclimation period: 14 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 0.16 °C
- Humidity( % ): 40 %
- Photoperiod(h dark / h light): 12 h dark / 12 h light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
A specified amount of the test material was weighed (not adjusted for purity), and transferred to a volumetric flask: which was first coated with the corn oil vehicle, using a series of vehicle rinses. Vehicle was then added in sufficient quantity to achieve the appropriate concentration. The flasks were inverted and manually shaken several times prior to stirring. The mixtures were stirred continuously for 5 minutes until homogenous using a magnetic stir plate and bars. The mixtures were prepared fresh daily and stirred prior to dispensing. A magnetic stir plate and bars were also utilized during dose administration to ensure adequate mixture. A total volume of 100 mL was prepared daily during the course of the study.

VEHICLE: CORN OIL
- Lot/batch no.: May 14 85
- Purity: 100 %

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: Presence of vaginal plug referred to as Day 0 of pregnancy

Duration of treatment / exposure:

10 d, from Day 6 to 15 of gestation, inclusive

Frequency of treatment:

Once daily

Duration of test:

25 d

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

30 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a range-finding study, the test dosage (1,000 mg/kg bw/day) was selected since it was anticipated to induce some degree of maternal toxicity but one which would not likely affect maternal survival.
- Rationale for animal assignment: Random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: From Day 0 through 20 of gestation

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, 6, 9, 12, 16 and 20.
- Mean body weight changes were calculated for each corresponding interval of gestation additionally for Day 6-16, 16-20 and 0-20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter

Statistics:

1. The fetal sex ratios were compared by the Chi-square test with Yates' correction factor.
2. The number of litters with malformations and developmental variations were compared by Fisher's Exact Test
3. The numbers of early and late resorptions, dead fetuses and postimplantation losses were compared by the Mann-Whitney U-test
4. Mean numbers of corpora lutea, total implantations, viable fetuses, mean fetal and maternal body weights and maternal body weight gain at each interval were analyzed by a one-way ANOVA and Dunnett's test

Indices:

- Fetal sex ratios, number of litters with malformations and developmental variations, numbers of early and late resorptions, dead fetuses and postimplantation losses
- Numbers of corpora lutea, total implantations and viable fetuses

Historical control data:

Yes, WlL historical control data appended in the report

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

salivation prior to and following dosing, urogenital matting and hair loss from various body surfaces

Mortality:

no mortality observed

Description (incidence):

Survival was 100% in the control and test item groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant decrease in mean body weight gain was noticed during gestation Day 6-16.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross internal morphological changes among the dams at the terminal sacrifice which could be considered treatment-induced. Sporadic findings such as renal infarct, white foci in the renal cortex or thickened gastic mucosa were observed in the test material groups and are not uncommon for rats of this strain and age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

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

Description (incidence and severity):

There were no biologically meaningful or statistically significant differences concerning the mean numbers of viable fetuses.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

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

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

All malformations observed are known to occur spontaneously in this strain of rat.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

All malformations observed are known to occur spontaneously in this strain of rat. A significant decrease occurred in the number of litters with fetuses having the 5th and 6th sternebrae unossified. This decrease is not considered biologically meaningful and is attributed to random occurrence.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

All malformations observed are known to occur spontaneously in this strain of rat

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no teratogenic or embryotoxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Jun 2018 - 25 Sep 2018

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:

2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 15-21 weeks old
- Weight at study initiation: between 3043 g and 4268 g
- Housing: housed individually in cages with perforated floors (Ebeco, Germany)
- Diet: ad libitum, Pelleted diet for rabbits (Global Diet 2030 from Harlan)
- Water: ad libitum, Municipal tap water
- Acclimation period: least 2 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 21
- Humidity (%): 38 to 97
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension; protected from light. If not used on the same day of preparation, the aliquots were stored in the refrigerator set to maintain 4°C for a maximum of 8 days. On the day of use, they were removed from the refrigerator and allowed to warm to room temperature for at least 30 minutes before dosing. Test item dosing formulations were kept at room temperature until dosing. If practically
possible, the dosing formulations and vehicle were continuously stirred until and during dosing.m Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: 1% Aqueous carboxymethyl cellulose with 0.5% Tween 80

Analytical verification of doses or concentrations:

yes

Remarks:

Acquity UPLC system

Details on analytical verification of doses or concentrations:

The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test item was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
The females arrived on Day 1-4 post-coitum (Day 0 post-coitum is defined as the day of successful mating).

Duration of treatment / exposure:

from Day 6 to Day 28 post-coitum

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of the tolerability study and dose range finder and in an attempt to produce graded responses to the test item. In the dose range finder, no dose-limiting toxicity was noted up to 400 mg/kg. However, in the tolerability study severe toxicity including mortality was observed at 750 mg/kg. Therefore, the dose levels selected in the current main teratology study were 0, 50, 150 and 450 mg/kg bw/day.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were individually weighed on Days 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured for Days 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum.

WATER CONSUMPTION:
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined:
All animals (including female no. 75 euthanized before planned necropsy) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. In addition, the gastro-intestinal tract was examined for signs of irritation/corrosion; the oesophagus was examined internally. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No tissues, except the uterus, were weighed. Each ovary and uterine horn of all animals was dissected and examined as quickly as possible.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all per litter ]

- Soft tissue examinations: Yes: [all per litter]

- Skeletal examinations: Yes: [all per litter]
Subsequently, the skeletal examination was done on all fetuses from all groups from Groups 1 and 4. Since no possible treatment related effects in the high dose group were seen, skeletal examination was not extended to the fetuses from the low and mid dose group.

- Head examinations: Yes: [half per litter]

Statistics:

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test. Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

see table 1

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicological relevant findings up to 450 mg/kg.
Reduced faeces production (up to a moderate degree) was observed in 14, 18, 19 and 18 females in the control, 50, 150 and 450 mg/kg groups, respectively, on several days during treatment. As all groups were affected (including the vehicle control group) and in the absence of a dose-related trend, no toxicological significance was attached to this observation. Incidental findings of note were red fluid on the manure tray in one high dose female, and lean appearance and/or piloerection in another high dose female. These findings were considered not to be toxicological relevant as they were observed in the vehicle control group at the same incidence (red fluid on the manure tray, lean appearance), were transient (lean appearance and piloerection) and all gravid females in this study had a normal pregnancy.
One female in Group 3 was noted with missing toes (two missing toes on the right foreleg and 1 missing toe on the left foreleg; taken from the study daybook). This finding was considered to be a congenital abnormality and thus unrelated to treatment with the test item. Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rabbits of this age and strain, and/or were observed in control females only. They were therefore of no toxicologically relevance.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatmentwith the test item.
One female from the high dose group had to be euthanized after 4 days of treatment. Immediately after dosing on post-coitum Day 9, she was observed with respiratory problems (gasping) and moribund. At necropsy, red foamy content of the trachea, several reddish foci in left caudal lobe of the lungs and grey-white discolouration of the left caudal lobe of the lungs were noted. Taken together, these findings were indicative of complications during the oral gavage procedure. Therefore, the preterm sacrifice of this female was considered unrelated to the test item.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight, body weight gain before and after correction for (gravid) uterus weight of treated Group 2, 3 and 4 females were unaffected by the treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after correction for body weight were recorded up to 450 mg/kg. In the high and mid dose group each, a trend towards slightly lower absolute and relative food consumption compared to the concurrent control group was noted from Days 6-15 and Days 12-15 post-coitum, respectively. As changes were only slight (reaching no statistical significance) and transient (complete recovery was seen from Days 15-18 post-coitum onwards), it was not considered as adverse.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. Moreover, the examination of the gastro-intestinal tract and the internal examination of the oesophagus did not reveal any signs of irritation and/or corrosion. The observation of missing toes in one Group 3 female was considered to be a congenital abnormality and thus unrelated to treatment with the test item. Remaining incidental findings among control and treated animals included watery-clear cysts of the oviducts, alopecia, scabs or an isolated wound. These findings are occasionally seen among rabbits used in these types of study, and in the absence of a dose relationship they were considered unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Mean pre-implantation loss was 3.6, 3.0, 3.0 and 10.5% for the control, 50, 150 and 450 mg/kg groups, respectively. The higher pre-implantation loss observed in the 450 mg/kg group could largely be attributed to one female (no. 76) who had 11 corpora lutea, but only 3 implantation sites, resulting in a pre-implantation loss of 72.7%. After excluding this female pre-implantation loss was 7.2%. As all values remained within the available historical control range the slightly higher pre-implantation loss in the high dose group was considered unrelated to treatment.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Overall, the number of pregnant females, corpora lutea and implantation sites, and pre-and post-implantation loss in the control and treatment groups were considered to be unaffected by treatment. Mean pre-implantation loss was 3.6, 3.0, 3.0 and 10.5% for the control, 50, 150 and 450 mg/kg groups, respectively. The higher pre-implantation loss observed in the 450 mg/kg group could largely be attributed to one female (no. 76) who had 11 corpora lutea, but only 3 implantation sites, resulting in a pre-implantation loss of 72.7%. After excluding this female pre-implantation loss was 7.2%. As all values remained within the available historical control range the slightly higher pre-implantation loss in the high dose group was considered unrelated to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: highest dose tested

Remarks on result:

other: Higher doses were not tolerated as two out of three females in the tolerability study had to be sacrificed in extremis on Days 4 and 7 of treatment at 750 mg/kg.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no toxicologically relevant effects on fetal body weights (both sexes) noted by treatment up to 450 mg/kg.
Mean combined (male and female) fetal body weights were 37.8, 38.2, 39.1 and 38.0 gram for the control, 50, 150 and 450 mg/kg groups, respectively.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to 450 mg/kg.
Mean sex ratios (males:females) were 46:54, 50:50, 44:56 and 56:44 for the control, 50, 150 and 450 mg/kg groups, respectively.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on litter size of any group.
Mean litter sizes were 10.4, 9.8, 10.1 and 9.3 fetuses/litter for the control, 50, 150 and 450 mg/kg groups, respectively. As all values remained within the available historical control range the slightly lower litter size in the high dose group was considered unrelated to treatment.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on external morphology following treatment up to 450 mg/kg/day.
Two external malformations were observed in this study. Two fetus of Groups 2 and 4, respectively, both had an open eye with relating findings of the eye/lens noted at soft tissue cephalic examination (i.e. eyelids not joined, lens attached to cornea and/or lens misshapen). Due to the single occurrence of this malformation in a low and high dose fetus, it was considered to be chance finding. A flexure of the carpal and/or tarsal was the other malformation and this occurred in the control group in one fetus (both tarsals, without apparent skeletal origin) and in late resorptions in two fetuses (one or both carpals, not skeletally examined). Because carpal and/or tarsal flexures were observed at the control level only, this was considered spontaneous in origin. External variations were not observed in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on skeletal morphology following treatment at 450 mg/kg/day.
The only fetus with a malformation was one fetus of the high dose, which had a vertebral anomaly with associated rib anomaly. A vertebral anomaly is the most common skeletal malformation among historical control fetuses and at the single occurrence noted in this study, it is considered spontaneous in origin. A statistical significant increase in the litter incidence of fetuses with caudal shift of pelvic girdle was observed in the high dose group. This variation occurred at an incidence of 21.5%
versus 8.7% per litter in the concurrent control group. Because the litter incidence of caudal shift of pelvic girdle was well within the historical control data range (mean 16.5% per litter; P5 – P95: 2.8 - 34.5% per litter), the higher incidence of this finding in Group 4 was considered to have occurred by chance and not to be toxicologically relevant. All other variations noted were not considered treatment related as they occurred infrequently, at frequencies that were within the range of available historical control data, or were observed in control fetuses only.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on visceral morphology following treatment up to 450 mg/kg/day. Visceral malformations occurred in 0 (0), 1 (1), 4 (3) and 3 (2) fetuses (litters) in the control, 50, 150 and 450 mg/kg groups, respectively.
In Group 4, the three malformed foetuses (out of two litters) had a malpositioned testis. This malformation was also noted in Group 3 fetus, but not in fetuses at the control and low dose levels. Nevertheless, because a malpositioned testis is one of the most common visceral malformations in historical control fetuses, this was considered not to be treatment related. The other malformed Group 3 fetuses all had a multiple cardiovascular malformation. As no cases occurred in Group 4 and none of
the individual malformations was uncommon among historical control fetuses these were considered not toxicologically relevant. The affected fetus in Group 2 had a diverticulum of the intestine, which at the single occurrence at the low dose was considered spontaneous in origin.
All variations noted were considered unrelated to treatment as they occurred in the absence of a dose-related trend, infrequently, in control fetuses only, and/or at frequencies that were within the range of available historical control data.

Key result

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Reference 3

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For read-across justification please refer to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: highest dose tested

Remarks on result:

other: Higher doses were not tolerated as two out of three females in the tolerability study had to be sacrificed in extremis on Days 4 and 7 of treatment at 750 mg/kg.

Key result

Abnormalities:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From June 28, 1984 to December 11, 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

other: Sprague Dawley COBS CD rats

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding laboratories, Inc., Portage, Michigan
- Age at study initiation: 13 wk
- Weight at study initiation: 225-297 g
- Housing: Individually housed in wire mesh cages suspended above cage board
- Diet: Purina certified rodent chow; ad libitum
- Water: Tap water; ad libitum
- Acclimation period: 14 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 0.16 °C
- Humidity (%): 40 %
- Photoperiod (h dark / h light): 12 h dark / 12 h light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing solution was prepared with corn oil using magnetic stir plate and bars


VEHICLE: CORN OIL
- Lot/batch no. (if required): May 14 85
- Purity: 100 %

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: Presence of vaginal plug referred to as Day 0 of pregnancy

Duration of treatment / exposure:

10 d, from Day 6 to 15 of gestation, inclusive

Frequency of treatment:

Once daily

Duration of test:

25 d

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

30 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a range-finding study, the test dosage (1000 mg/kg/d) was selected since it was anticipated to induce some degree of maternal toxicity but one which would not likely affect maternal survival.
- Rationale for animal assignment: Random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: From Day 0 through 20 of gestation


BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, 6, 9, 12, 16 and 20
- Mean body weight changes were calculated for each corresponding interval of gestation additionally for Day 6-16, 16-20
and 0-20


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter

Statistics:

1. The fetal sex ratios were compared by the Chi-square test with Yates' correction factor.
2. The number of litters with malformations and developmental variations were compared by Fisher's Exact Test
3. The numbers of early and late resorptions, dead fetuses and postimplantation losses were compared by the Mann-Whitney U-test
4. Mean numbers of corpora lutea, total implantations, viable fetuses, mean fetal and maternal body weights, and maternal body weight gain at each interval were analyzed by a one-way ANOVA and Dunnett's test

Indices:

- Fetal sex ratios, number of litters with malformations and developmental variations, numbers of early and late resorptions, dead fetuses and postimplantation losses
- Numbers of corpora lutea, total implantations and viable fetuses

Historical control data:

Yes, WlL historical control data appended in the report

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
- Two rats died due to an intubation error
- Slight decrease in mean body weight gain was observed during gestation Day 12-16
- Significant decrease in mean body weight gain was noticed during gestation Day 6-16
- Clinical signs of toxicity such as salivation prior to and following dosing; urogenital matting and hair loss from various body surfaces were observed

Dose descriptor:

dose level:

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

clinical signs

mortality

Remarks on result:

not determinable

Remarks:

no NOAEL determinable

Abnormalities:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Two malformed fetuses occurred and are known to occur spontaneously in this strain of rat
- No statistically significant or biologically meaningful embyrotoxic/teratogenic effects occurred in the treatment group

Key result

Dose descriptor:

NOAEL

Remarks:

embryotoxicity

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: highest dose tested

Abnormalities:

not specified

Key result

Developmental effects observed:

no

Reference 5

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

For read-across justification please refer to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

dose level:

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

clinical signs

mortality

Remarks on result:

not determinable

Remarks:

no NOAEL determinable

Abnormalities:

not specified

Key result

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

embryotoxicity

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: highest dose tested

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

no

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

Quality of whole database:

similar to OECD TG 414

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 study on Rats:

A study designed to screen potential maternal, embryotoxic and teratogenic effect of the test item in the rat was performed (Cytec, 1984). The study was conducted equivalent or similar to OECD Guideline No. 414. The substance was administered orally, admixed in corn oil, to one group of 30 bred rats at a dosage level of 1,000 mg/kg bw/day, from gestation Day 6 through 15. Control group consisting of 30 bred rats received the corn oil vehicle on a comparable regimen, at 10 mL/kg. Throughout gestation, all rats were observed twice daily for signs of toxicity. Body weights were recorded at appropriate intervals. All surviving animals were sacrificed on gestation Day 20 for the scheduled Cesarean section. Fetuses were sexed, weighed and examined for external, skeletal and soft tissue anomalies and developmental variations. Clinical signs of toxicity such as salivation prior to and following dosing, urogenital mating and hair loss from various body surfaces were observed. Mean body weight gains were significantly reduced over the entire treatment period. No embryotoxic effects were apparent and developmental variations were not remarkably different between the control and test material group. No statistically significant or biologically meaningful differences occurred between the control and treatment regarding the occurrence of malformations. In conclusion, the substance produced observable maternal toxicity at a dose level of 1000 mg/kg bw/day but did not induce a teratogenic or embryotoxic effect in rats.

 

Prenatal developmental study on Rabbits:

The objectives of this study (ReachCentrum, 2019) were to determine the potential of the read-across substance (CAS 42978-66-5) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels in this study were selected to be 0, 50, 150, 450 mg/kg bw/day, based on the results of the dose range finder and tolerability study. In the dose range finder, no dose-limiting toxicity was noted up to 400 mg/kg. However, in the tolerability study severe toxicity was observed at 750 mg/kg. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents, and maternal pregnancy data. In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations. Formulation analyses confirmed that formulations of test item in 1% aqueous carboxymethyl cellulose with 0.5% Tween 80 were prepared accurately and homogenously. No maternal toxicity was observed in the 50, 150 and 450 mg/kg groups. No developmental toxicity was observed in the 50, 150 and 450 mg/kg bw/d groups. In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) of at least 450 mg/kg was established. Higher doses were not tolerated as two out of three females in the tolerability study had to be sacrificed in extremis on Days 4 and 7 of treatment at 750 mg/kg bw/d.

 

In order to support the read-across approach there is an additional prenatal developmental toxicity studies on rats available:

 

Rat:

An additional supporting study with the structural analogue substance CAS 28961-43-5 is available to screen potential maternal, embryotoxic and teratogenic effects in rats (Cytec, 1984). The study was conducted equivalent or similar to OECD Guideline No. 414 in compliance with Good Laboratory Practices. The test substance was administered orally, admixed in corn oil, to one group of 30 bred rats at a dosage level of 1000 mg/kg bw/day, from gestation Day 6 through 15. Control group consisting of 30 bred rats received the corn oil vehicle on a comparable regimen, at 10 mL/kg. Throughout gestation, all rats were observed twice daily for signs of toxicity. Body weights were recorded at appropriate intervals. All surviving animals were sacrificed on gestation Day 20 for the scheduled Cesarean section. Fetuses were sexed, weighed and examined for external, skeletal and soft tissue anomalies and developmental variations. Clinical signs of toxicity such as salivation prior to and following dosing, urogenital matting and hair loss from various body surfaces were observed. Mean body weight gains were significantly reduced over the entire treatment period. No embryotoxic effects were apparent. Developmental variations were not remarkably different between the control and test material group. No statistically significant or biologically meaningful differences occurred between the control and treatment regarding the occurrence of malformations.  In conclusion, the substance produced substantial maternal toxicity at a dose level of 1000 mg/kg bw/day but did not induce a teratogenic or embryotoxic effect in rats.

Conclusion

Based on the available prenatal developmental toxicity studies with the target and the source substances on rats and rabbits it is concluded that the target substance is not expected to induce a teratogenic or embryotoxic effect. Impairment of prenatal development was observed neither for the target nor for the source substances up to and including dose levels inducing maternal toxicity. Moreover, in all cases the tolerability of the source and the target substances was limited by local effects in the (fore)stomach rather than any systemic signs of toxicity.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available information are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available experimental information,the test substance is not classified for toxicity to reproduction or developmental toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.