Glycerol, propoxylated, esters with acrylic acid

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Glycerol, propoxylated, esters with acrylic acid

EC Number:

500-114-5

EC Name:

Glycerol, propoxylated, esters with acrylic acid

Cas Number:

52408-84-1

Molecular formula:

(C3H6O)m(C3H6O)n(C3H6O)oC12H14O6

IUPAC Name:

Poly[oxy(methyl-1,2-ethanediyl)], .alpha.,.alpha.',.alpha.''-1,2,3- propanetriyltris[.omega.-[(1-oxo-2-propenyl)oxy]]-

Test material form:

liquid

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

375 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

1

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

2

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

3

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

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no