4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

In the Extended one-generation reproduction study (OECD 443, 2019) and in the combined repeated dose and reproduction / developmental screening test (OECD 422, 2010), no adverse effect in the on reproduction parameters were observed at the maximal doses of 200 and 900 mg/kg/day respectively. However some signs of toxicity (not adverse) were observed in the studies.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

15 January 2010 to 2 June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 310 to 366 g for males and 207 to 244 g for females
- Housing: up to 5 during pre-mating for all animals and after mating for males, individually with litter for females during gestation and littering.
- Diet (e.g. ad libitum): standard rodent diet (SDS VRF1 Certified) ad libitum, except overnight before routine blood sampling for Toxicity subgroup animals.
- Water (e.g. ad libitum): potable water taken from the public supply, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23
- Humidity (%): 40 to 70
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: 20 January 2010 To: 13 March 2010

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: approximately 50% of the final volume of propylene glycol was added to the test material. The formulation was warmed in a water bath at approximately 50°C and stirred with a spatula until all the test material had dissolved and more propylene glycol was added to make up the required volume. Then, the formulation was returned to the container and mixed using a magnetic stirrer until homogeneous. If necessary, the dose was warmed again to aid homogenisation.
Initially all formulations were prepared freshly on the day of use and used within two hours of completion of preparation. However, following
confirmation of the results from a homogeneity and stability study (Huntingdon Life Sciences Study No. RAJ0005) formulations were prepared freshly each week and stored refrigerated (approximately 4°C) until required for use.

VEHICLE
- Concentration in vehicle: 20, 60 and 180 mg/mL
- Amount of vehicle (if gavage): constant dosage-volume of 5 mL/kg bw/day

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in the first and last weeks of the dosing period were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL accurately weighed) from all groups. Two assays from each group were analysed. The analytical method involved dissolution and dilution using acetonitrile followed by reverse phase high performance liquid chromatographic analysis with ultra-violet detection (230 nm). The mean concentrations of CAS 55818-57-0 in test formulations analysed for the study were within applied limits +10%/-15% of nominal concentrations, confirming accurate formulation, with the exception of one result, Week 1, Day 1, Group 2, 20 mg/mL which was 20.5% below nominal. Additional samples taken from the reformulated Week 1, Day 5, Group 2 formulation were therefore analysed and the achieved concentration was 1.5% above nominal, confirming the accuracy of formulation.

Duration of treatment / exposure:

Five consecutive weeks for Toxicity subgroup males and females and Reproductive subgroup males. Reproductive subgroup females were treated daily for two weeks prior to pairing, throughout pairing and up until the day prior to termination on Day 7 of lactation. Offspring were not dosed.

Frequency of treatment:

Once a day, 7 days a week

Details on study schedule:

None

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

Toxicity subgroup: 5 males and 5 females/dose
Reproductive subgroup: 5 males and 10 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: in a preliminary study (Huntingdon Life Sciences Study Number: RAJ0001), dose levels of 80, 250, 750 and 1000 mg/kg bw/day were employed and there was a suggestion of reduced bodyweight gain among males that received 1000 mg/kg bw/day. There was no evidence of an effect of treatment on clinical condition, food consumption, oestrous cycles or organ weights at any dose level investigated, and no treatment-related macroscopic abnormalities were observed. For this study, treatment would continue for approximately 5 weeks and females would be pregnant, therefore in recognition of the potential for reduced bodyweight gain, the high dose level was set at 900 mg/kg bw/day, the low dose of 100 mg/kg bw/day was chosen as an anticipated No Observed Adverse Effect Level (NOAEL), and the intermediate dose of 300 mg/kg bw/day at a logarithmic interval between the low and high doses.

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of ill-health amongst the occupant(s). During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS:
Detailed observations were recorded in relation to dose administration. For the Toxicity subgroup animals and for Reproductive subgroup males these were recorded daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and on one occasion during Week 5. For Reproductive subgroup females these were recorded daily during the first week of treatment, twice weekly during Week 2 of treatment, on Days 0, 4, 7, 11, 14 and 20 after mating and Day 4 of lactation.

Before treatment commenced and during each week of treatment, detailed physical examination and arena observations were performed on each animal (physical condition and behaviour during handling with particular attention to possible signs of neurotoxicity). For the Reproductive subgroup females during the post-mating period, these observations were conducted on Days 0, 7, 14 and 20 after mating and on Days 1 and 7 of lactation.

BODY WEIGHT:
Toxicity subgroup males and females and Reproductive subgroup males were weighed weekly throughout the study. Reproductive subgroup females were weighed weekly until mating was detected, on Days 0, 7, 14 and 20 after mating and on Days 1, 4, 6 and 7 of lactation.

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded weekly for all animals for the first two weeks of treatment. Food consumption continued to be recorded weekly for the remaining three weeks of treatment for Toxicity subgroup females. Food consumption was not recorded during Week 3 of treatment for Toxicity subgroup males or Reproductive subgroup males as the animals were in pairing with the Reproductive subgroup females during this period. Food consumption was recorded, however, during Weeks 4 and 5 of treatment for these males.
For each Reproductive subgroup female, the weight of food supplied, that remaining and an estimate of any spilled was recorded for the periods Days 0-6, 7-13 and 14-19 after mating and Days 1-3 and 4-6 of lactation.

WATER CONSUMPTION:
Fluid intake was assessed by daily visual observation.

OTHER:
- SENSORY REACTIVITY:
Sensory reactivity and grip strength assessments were performed (before dosing) on all Toxicity subgroup animals during Week 5 of treatment. The following measurements, reflexes and responses were recorded: approach response, touch response, auditory startle reflex, tail pinch response and grip strength.

- MOTOR ACTIVITY: During Week 5 of treatment (before dosing), the motor activity of each Toxicity subgroup animal was measured using a Rodent Activity Monitoring System (Version 2.0.3). Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total).

- HAEMATOLOGY:
During Week 5 of treatment (after sensory reactivity, grip strength and motor activity assessments), blood samples were obtained from all Toxicity subgroup animals after overnight withdrawal of food. Animals were held under light general anaesthesia induced by isoflurane and blood samples were withdrawn from the sublingual vein. The following were measured using a Bayer Advia 120 haematology analyser: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Reticulocyte count (Retic), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total leucocyte count (WBC), Differential leucocyte count (Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B)
Monocytes (M), Large unstained cells (LUC)) and Platelet count (Plt). The most common morphological changes, anisocytosis, micro/macrocytosis and hypo/hyperchromasia were recorded. Prothrombin time (PT) (using an ACL 3000 Plus analyser and IL PT-Fibrinogen reagent) and Activated partial thromboplastin time (APTT) (using an ACL 3000 Plus Analyser and IL APTT reagent) were also measured.

- BLOOD CHEMISTRY:
At the same time and using the same animals as for peripheral haematology, the plasma was examined using a Roche P Modular Analyser for: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin (Bili), Bile Acids (BIAC), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot) and Albumin (Alb) (by chemical assay).

Oestrous cyclicity (parental animals):

For 15 days before pairing (including the day of pairing), daily vaginal smears (dry) were taken from all Reproductive subgroup females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed.

Sperm parameters (parental animals):

No data

Litter observations:

PARAMETERS EXAMINED:
All litters were examined at approximately 24 hours after birth and then daily thereafter for clinical signs (evidence of ill health or reaction to treatment), litter size (mortality and consequent changes in litter size from Days 1-7 of age), sex ratio of each litter (recorded on Days 1, 4 and 7 of age) and individual bodyweight (recorded on Days 1, 4 and 7 of age).

GROSS EXAMINATION OF PUPS:
All live pups were sacrificed on day 7 and were subject to a detailed necropsy. Missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before Day 7 of age were examined.

Postmortem examinations (parental animals):

SACRIFICE:
Toxicity subgroup males and females and Reproductive subgroup males were killed in Week 6 after completion of the Week 5 investigations.
Reproductive subgroup females were killed on Day 7 of lactation.

GROSS NECROPSY:
All animals were subject to a detailed necropsy. For Reproductive subgroup females, the number of uterine implantation sites was also recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS:
The tissues indicated in Table 1 were weighed.
In the Toxicity subgroup, the following organs were fixed for histopathology: Adrenal glands, Peyer’s patch, Brain, Pituitary, Caecum, Prostate, Colon, Rectum, Duodenum, Sciatic nerves, Epididymides (L&R), Seminal vesicles and coagulation gland, Heart, Spinal cord, Ileum, Spleen, Jejunum, Sternum with marrow, Kidneys, Stomach, Liver, Testes (L&R), Lungs, Thymus, Lymph nodes (left axillary and mesenteric), Thyroid with parathyroids, Trachea, Mammary area (caudal), Urinary bladder, Oesophagus, Uterus with cervix and oviducts, Ovaries (L&R) and Vagina.
In the Reproductive subgroup, the following organs were fixed for histopathology: Mammary area (caudal), Testes (L&R), Ovaries (L&R), Uterus with cervix and oviducts, Pituitary, Vagina and Prostate. Samples of any abnormal tissues were also retained and processed for examination.
All tissues preserved for examination were examined for all Toxicity subgroup and Reproductive subgroup animals of Control group and 900 mg/kg bw/day group. In addition, based on the results of the initial assessment, the lungs and kidneys were examined from all Toxicity subgroup males in the 100 and 300 mg/kg bw/day groups.

Postmortem examinations (offspring):

All live pups were sacrificed on day 7 and were subject to a detailed necropsy. Missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before Day 7 of age were also examined.

Statistics:

The following sequence of statistical tests was used for grip strength, motor activity, bodyweight, food consumption, organ weight, litter size and survival indices and clinical pathology data: 1) a parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead. 2) a non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test (Steel 1959) was performed instead.
For organ weight data, analysis of covariance was performed. Sex ratio were analysed by Wald chi-square test.
For gestation length an exact two-tailed Linear-by-linear test (Cytel 1995), with equally spaced scores, was applied to all groups.

Reproductive indices:

Percentage mating : Number animals mating / Animals paired × 100
Conception rate (%) : Number animals achieving pregnancy / Animals mated × 100
Fertility index (%) : Number animals achieving pregnancy / Animals pairing × 100

Offspring viability indices:

Gestation index (%) : Number of live litters born / Number pregnant × 100
Post - implantation survival index (%) : Total number offspring born / Total number uterine implantation sites × 100
Live birth index (%) : Number live offspring on Day 1 after littering / Total number of offspring born × 100
Viability index (%) : Number live offspring on Day 4 after littering / Number live offspring on Day 1 after littering × 100
Lactation index (%) : Number live offspring on Day 7 after littering / Number live offspring on Day 1 after littering × 100

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One Reproductive subgroup male receiving 100 mg/kg bw/day was found dead prior to dose administration on Day 34 of the study. Ante mortem signs for this animal were restricted to noisy respiration (rales) on the previous day and there were no macroscopic abnormalities detected. In the absence of any other mortalities in this or higher dose groups, it was considered incidental and unrelated to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 900 mg/kg/day, mean bodyweight gain was statistically significantly lower than Control during the first week of treatment (67% of Control). During the second week of treatment, the weight gain of these males remained slightly (but not significantly) lower than Control. Thereafter, mean weight gain was essentially similar to Control and overall mean gain during the treatment period (Week 0 to Week 5) was 81% of Control. The mean bodyweight gain of males receiving 300 or 100 mg/kg bw/day was considered unaffected by treatment.
Mean bodyweight gain of females in all dose groups was considered unaffected by treatment throughout the study. It was noted that during the first two weeks of treatment, mean weight gain in all treated groups was lower than Control, and between Weeks 2 and 5 the weight gain of Toxicity subgroup females was higher than Control, with all differences attaining statistical significance. There was, however, no dose response apparent, and therefore these differences were considered to be of no toxicological importance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean food intake of males and females in all dose groups was considered unaffected by treatment throughout the study.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

see section 7.5.1

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

see section 7.5.1

Urinalysis findings:

no effects observed

Description (incidence and severity):

see section 7.5.1

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

see section 7.5.1

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test article related changes were observed in any of the organs microscopically examined from Toxicity and Reproductive subgroup animals.
Seminiferous tubules of all males of both the Toxicity and Reproductive subgroups were evaluated with respect to their stage in the spermatogenic cycle and integrity of various cell types present within the different stages. No cell or stage specific abnormalities were noted.
During initial microscopical examination, lungs from three Toxicity subgroup males receiving 900 mg/kg bw/day had minimal aggregations of alveolar macrophages. In addition, the kidney from one male in this group had minimal multiple cortical scarring, minimal focal cortical tubular basophilia and cortical tubular dilatation. To assess the toxicological significance of this finding, a trackdown was carried out on lungs and kidney from Toxicity and Reproductive subgroup males given 100 or 300 mg/kg bw/day. Subsequent microscopical examination showed no dose dependent trend in severity or incidence level for these findings and hence they were considered as incidental.
The incidence and distribution of all the other findings were consistent with the common background for animals of this age at these laboratories.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Oestrous cycle length, pre-coital interval, mating performance and fertility and gestation length of the Reproductive subgroup females was unaffected.

Dose descriptor:

NOAEL

Effect level:

> 900 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed for F1 offspring that were considered to be related to parental treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no effect of parental treatment at any dose level investigated on the mean number of implantation sites or litter size. Post-natal survival was unaffected by treatment, and the percentage of males in the litters in all groups was consistent between Days 1 and 7 of age, indicating that there was no preferential mortality of either sex.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no evidence of an adverse effect of parental treatment at any dose level investigated on mean offspring bodyweights at birth, or on subsequent weight gain to Day 7 of age.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

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 macroscopic abnormalities detected among the offspring that died during the early post-natal period, or at scheduled termination on Day 7 of age that were attributable to parental treatment.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

There were no clinical signs observed for F1 offspring that were considered to be related to parental treatment with test substance, and offspring survival and growth from birth to Day 7 of age was unaffected by treatment.
There were no macroscopic abnormalities detected among the offspring that died during the early post-natal period, or at scheduled termination on Day 7 of age that were attributable to parental treatment with test substance.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 900 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

not specified

Conclusions:

Based on the results of this study, it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity in the CD rat following 5 weeks of treatment was higher than 900 mg/kg bw/day. The NOAEL for the reproductive/developmental toxicity screening test within the scope of this study was also concluded to be higher than 900 mg/kg bw/day.

Executive summary:

In a GLP study conducted according to OECD guideline 422, three groups each comprising five male and five female rats for the Toxicity subgroup and five male and ten female rats for the Reproductive subgroup received 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid at doses of 100, 300 or 900 mg/kg bw/day. Toxicity subgroup males and females and Reproductive subgroup males were treated daily for five consecutive weeks. Reproductive subgroup females were treated daily for two weeks before pairing, throughout pairing, gestation and lactation until the day prior to termination on Day 7 of lactation. A similarly constituted Control group received the vehicle, propylene glycol, at the same volume-dose.

During the study, data was recorded on clinical condition, performance under detailed physical and arena examination, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, water consumption (visual), haematology, blood chemistry, oestrous cycles, mating performance and fertility and gestation length. Organ weight, macroscopic and microscopic pathology investigations were undertaken in the adults. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken.

There was only one mortality during the course of the study, therefore considered unrelated to treatment. There were no signs observed among treated males and females at routine physical examination or during the arena observations that were considered to be related to treatment. There was no effect of treatment on the food consumption, sensory reactivity findings, grip strength values and motor activity in males and females throughout the study.

At 900 mg/kg bw/day, mean bodyweight gain of males was slightly lower than Control during the first week of treatment. During the second week, weight gain for these males remained slightly (but not significantly) lower than Control, but thereafter, mean weight gain was essentially similar to Control in all treated groups, and the bodyweight gain of females during gestation and lactation was unaffected by treatment at all dose levels investigated.

Haematological assessment revealed slightly prolonged prothrombin time among males and females receiving 900 or 300 mg/kg bw/day. Biochemical changes in the blood plasma comprised high total bilirubin and total bile acid concentrations in males receiving 900 or 300 mg/kg bw/day and females receiving 900 mg/kg bw/day, and high cholesterol concentrations in males and females in all treated groups. A dose-related trend was evident for all of these haematological and biochemical parameters.

There was no clear evidence of an adverse effect of treatment on mean organ weights among all animals at scheduled termination. There were no macroscopic abnormalities and no test substance-related lesions at microscopic examination.

Oestrous cycle length, pre-coital interval, mating performance and fertility and gestation length of the Reproductive subgroup females were unaffected by treatment. There were no clinical signs observed for F1 offspring that were considered to be related to parental treatment and offspring survival and growth from birth to Day 7 of age was unaffected by treatment. There were no macroscopic abnormalities detected among the offspring that died during the early post-natal period, or at scheduled termination on Day 7 of age that were attributable to parental treatment.

Based on the results of this study, it was concluded that the NOAEL for systemic toxicity in the CD rat following 5 weeks of treatment was higher than 900 mg/kg bw/day. The NOAEL for the reproductive/developmental toxicity screening test within the scope of this study was also concluded to be higher than 900 mg/kg bw/day.

Reference 2

Endpoint:

extended one-generation reproductive toxicity - with both developmental neuro- and immunotoxicity (Cohorts 1A, 1B without extension, 2A, 2B, and 3)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2018 - September 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:

June 2018

Deviations:

no

Principles of method if other than guideline:

OECD guidance document supporting OECD test guideline 443 on the extended one-generation reproductive toxicity test, No. 151, July 2013.

GLP compliance:

yes (incl. QA statement)

Remarks:

d.d. 22 January 2018

Limit test:

no

Justification for study design:

The design of this study was based on the final decision on a compliance check of DGEBADA by ECHA (Decision no. CCH-D-2114375349-37-01/F, date 24 Oct 2017).
The European authorities required an “ Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: EU B.56./OECD TG 443) in Wistar rats, oral route with the registered substance specified as follows: Ten weeks premating exposure duration for the parental (P0) generation; Dose level setting shall aim to induce some toxicity at the highest dose level; Cohort 1A (Reproductive toxicity); Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation; Cohorts 2A and 2B (Developmental neurotoxicity); and Cohort 3 (Developmental immunotoxicity).
The oral gavage route of administration was selected as this allows for a more accurate dosing than dietary administration, and since previous toxicity studies conducted with this test item were also conducted using this route of administration.

Species:

rat

Strain:

Wistar

Remarks:

(Wistar Han rats)

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/neurological/immunological internal data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive, neurological and immunological toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 6-7 wks
- Weight at study initiation: (P) Males: 133-173 g; Females: 112-147 g

- 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; height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (type III; height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, type IV; height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (type III, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (type III, height 18 cm). Pups were housed with the dam until termination (unscheduled deaths, spares, and pups of Cohort 2B) or until weaning on PND 21 (Cohorts 1A, 1B, 2A and 3).
During locomotor activity monitoring, F1- Cohort 2A animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water for a maximum of 2 hours.
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted by study procedures/activities.
Animals were separated during designated procedures/activities.

- Diet: ad libitum
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, F1- Cohort 2A animals had no access to food for a maximum of 2 hours.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

- Water: ad libitum
Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, F1- Cohort 2A animals had no access to water for a maximum of 2 hours.
Periodic analysis of the water was performed. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22
- Humidity (%): 43-67
- Air changes (per hr): Ten or greater
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 25 September 2018 To: 16 April 2019

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG400

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. Formulations were heated to a maximum temperature of 44°C for at least 30 minutes to a maximum of approximately 8 hours to obtain visual homogeneity. The dosing formulations were prepared in daily portions, maximally 7 days prior to dosing. If these were dosed on the day of formulating, they were dosed within 6 hours after adding the vehicle to the test item. Formulations were released for dosing when they had obtained a temperature of 40°C or lower. If not dosed on the day of formulating, the formulations were stored in the refrigerator and removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. From 16 Jan 2019 onwards, formulations that had solidified in the refrigerator were heated to maximally 30°C for maximally 2 hours in a water bath in order to accelerate liquefying the formulations.
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.
An additional formulation was prepared for the Group 2 F1 concentration in Week 21 and 25 of the study for intended stability measurements (these formulations were used for sampling only).

VEHICLE
- Justification for use and choice of vehicle: Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. In these trial preparations, water (Elix), 1% aqueous carboxymethyl cellulose, propylene glycol and polyethylene glycol 400 were tested. From these trial preparations, polyethylene glycol 400 was selected as a suitable vehicle. Polyethylene glycol 400 is a commonly used vehicle for repeated toxicity studies in rats, and is regarded not harmful for rats. Moreover, polyethylene glycol (300) was also used in a 90-day toxicity study with DGEBADA in Wistar Han rats according to OECD TG 408 animals.
- Concentration in vehicle: F0 animals 8, 20 and 40 mg/mL; F1 animals 2, 5 and 10 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw

A positive control item was used for the TDAR assay in cohort 3
- vehicle: Sterile saline solution (0.9% NaCl)
Each day of dosing, a dose formulation of 1.5 mg/mL Cyclophosphamide was freshly prepared by dissolving cyclophosphamide monohydrate in sterile saline solution. No adjustment was made for specific gravity of the vehicle and cyclophosphamide monohydrate. No correction was made for the purity/composition of cyclophosphamide monohydrate.
Cyclophosphamide formulations were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Formulations were dosed within 6 hours after adding the vehicle to the test item.
Dosing formulations were kept at room temperature until dosing. The dosing formulations were gently inverted until and during dosing.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.
- Proof of pregnancy: sperm in vaginal smear or vaginal plug referred to as day 0 of pregnancy
- Further matings after unsuccessful attempt: no
- After successful mating each pregnant female was caged: Individually
- Any other deviations from standard protocol:
Detection of mating was not confirmed in first instance for one Group 2 and one Group 3 female. Evidence of mating was obtained indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continuation of di-estrus during the mating in these females. The mating date of these animals was estimated at 21 days prior to the actual delivery date This day was designated Day 0 post-coitum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20153909) demonstrated that the test item is stable in the vehicle for at least 6 hours at room temperature, at least 8 days in the refrigerator and for at least 3 weeks in the freezer (= -15°C) when prepared and stored under the same conditions at concentrations bracketing those used in the present study. In order to cover the time period required and concentrations used for dosing all F0 and F1 animals in the study, an additional stability measurement was required over 24 hours at room temperature under normal laboratory light conditions.

Concentration, homogeneity and stability analysis
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Analyses were performed using a validated analytical procedure.

The following dose formulation samples were collected:
Week 1, 2, 3, 5, 9, 13 and 15: concentration analyses (all groups), homogeneity analysis (groups 2 and 4)
Week 21: concentration analyses (all groups + group 2 (F1)), homogeneity analysis (groups 2 and 4 + group 2 (F1)), stability assessment (group 4 (F0) + group 2 (F1))
Week 25: concentration analyses (all groups + group 2 (F1)), homogeneity analysis (groups 2 and 4 + group 2 (F1)), stability assessment (group 2 (F1))

It was noted that the concentration of the Group 2 formulations prepared for the Week 21 analyses was well outside the acceptance criterion due to a weighing error during formulation preparation. It was therefore decided not to analyze stability of this formulation. Instead, new formulations were prepared as part of the Week 25 analyses, including stability analyses. Accuracy and homogeneity was also determined from the Group 2 formulation with a concentration that was used for dosing the F1 animals between PND 7 and 20. The homogeneity data served as t=0 for the stability measurement.

The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results.

Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was =10%.
Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation.

Duration of treatment / exposure:

(P) Males: 12 weeks (including 10 weeks pre-mating)
(P) Females: 13-16 weeks; 10 weeks before mating, up to 2 weeks during mating, approximately 3 weeks during resulting pregnancies, one week through weaning of their F1 offspring.
(F1) Cohort 1A: 12-13 weeks; direct dosing of pups on PND 7-20 and post-weaning for 10-11 weeks
(F1) Cohort 1B: 13-15 weeks; direct dosing of pups on PND 7-20 and post-weaning for 11-13 weeks
(F1) Cohort 2A: 10-12 weeks; direct dosing of pups on PND 7-20 and post-weaning for 8-10 weeks
(F1) Cohort 2B: 2 weeks; direct dosing of pups on PND 7-20, except for animals that were necropsied on PND 22 which were also dosed on PND 21 using dose levels for PND 7-20
(F1) Cohort 3: 6-8 weeks; direct dosing of pups on PND 7-20 and post-weaning for 4-6 weeks
F1 animals allocated to the positive control group (group 5) were not directly dosed with the test item.

Frequency of treatment:

once daily, 7 days a week

Details on study schedule:

Cohort 1B was not extended to produce a F2 generation

Dose / conc.:

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

Remarks:

Group 2.
F1 animals directly dosed between PND 7 and 20 received 10 mg/kg bw/day (actual dose received)

Dose / conc.:

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

Remarks:

Group 3.
F1 animals directly dosed between PND 7 and 20 received 25 mg/kg bw/day (actual dose received)

Dose / conc.:

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

Remarks:

Group 4.
F1 animals directly dosed between PND 7 and 20 received 50 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

F0: 25
F1 cohort 1A: 20
F1 cohort 1B: 20
F1 cohort 2A: 10
F1 cohort 2B: 10
F1 cohort 3: 10 males and 9 females
F1 positive control: 10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels for F0 animals and for F1 animals post-weaning were selected based on the results of previously conducted (repeated and reproduction) toxicity studies with oral exposure of DGEBADA in rats as detailed below.
In a reproductive/developmental toxicity screening study in Sprague-Dawley rats according to OECD TG 422, animals were dosed at 100, 300 and 900 mg/kg/day by oral gavage (vehicle: propylene glycol). A NOAEL of 900 mg/kg/day was established for parental and reproductive/developmental effects, in absence of adverse effect observed at this maximal dose.
In a 90-day toxicity study in Wistar Han rats according to OECD TG 408 animals were dosed at 100, 300 and 1000 mg/kg/day by oral gavage (vehicle PEG 300). Males at 1000 mg/kg/day had lower body weights (22% lower weight gain than controls) and locomotor activity of males was statistically significantly lower at 300 and 1000 mg/kg/day without concurrent changes in clinical observations or other functional observation parameters. Various clinical pathology changes were noted at 300 and 1000 mg/kg/day including higher cholesterol and phospholipid values. Lower prostate weights were recorded at all dose levels, but occurred in the absence of supportive histopathological lesions and organ weights of other sex organs were normal. Kidney weights were higher at 300 and 1000 mg/kg/day in males. Reduced sperm motility was noted at all dose levels. However, there were no effects on sperm counts and more than 50% of the sperm cells showed progressive motility (mean progressive motility showed no clear dose-related trend). Histopathological changes were noted at 1000 mg/kg/day, consisting of hepatocellular hypertrophy and necrosis, decreased lymphocyte counts in mesenteric lymph nodes and multifocal hypertrophy in the pituitary gland in individual animals. Based on the results of this 90-day repeated toxicity study, a NOAEL for systemic toxicity was below the lowest dose level of 100 mg/kg bw/day of DGEBADA.
In a developmental toxicity study in Wistar Han rats according to OECD TG 414, animals were dosed at 100, 300 and 1000 mg/kg/day by daily oral gavage (vehicle PEG 300). A NOAEL of 1000 mg/kg/day was derived for maternal toxicity and developmental toxicity, in absence of adverse effect observed at this maximal dose.
Based on adverse effects observed in the 90-day repeated toxicity in which no NOAEL was established (LOAEL = 100 mg/kg/day), dose levels were selected to be 40, 100 and 200 mg/kg/day in an attempt to produce graded responses to the test item. The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level was expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
The dose levels for F1 animals between PND 7 and 20 were selected based on the dose levels used for F0 animals. Available F0 in-life data indicated that treatment up to 200 mg/kg/day was well tolerated. However, pups may be more vulnerable to treatment than F0 animals given that physiological processes and systems (such as the blood-brain barrier and metabolic processes) of these pups are still developing. Therefore, in order to avoid potential overdosing, the highest dose selected for F1 animals (50 mg/kg/day) was 25% of the high dose administered to F0 animals (200 mg/kg bw/day). The low and mid dose levels for F1 animals (10 and 25 mg/kg bw/day, respectively) were selected using the same relative dose level spacing as for F0 animals.


- Rationale for animal assignment:
F0 animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.
On PND 4, eight pups from each litter of equal sex distribution (if possible) were selected to reduce variability among the litters. The non-selected pups were culled on PND 4. To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.
On PND 21, pups from available litters per group were selected and assigned into cohorts.

Positive control:

F1 positive control animals were treated with cyclophosphamide 2 to 4 hours prior to immunization with KLH (Group 5). Cyclophosphamide was administered via intraperitoneal injection using a disposable needle and syringe once daily on five consecutive days prior to necropsy (i.e. starting between PND 48-54), approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Formulations were gently inverted prior to dosing. The dose volume for each animal was based on body weight measurement on Day 1 of treatment.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- General health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were conducted in a standard arena once before the first administration of the test item and at weekly intervals during the treatment period.

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:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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: Yes
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight with a maximum of 24 hours before blood sampling, but water was available.
- How many animals: 10 selected animals/sex/group F0-animals
- Parameters checked: according to OECD TG 443

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. on the day of scheduled necropsy
- Animals fasted: Yes, overnight with a maximum of 24 hours before blood sampling, but water was available.
- How many animals: 10 selected animals/sex/group F0-animals
- Parameters checked: according to OECD TG 443

URINALYSIS: Yes
- Time schedule for collection of urine: Overnight (15-20 hrs)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: according to OECD TG 443

THYROID HORMONE
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. on the day of scheduled necropsy
- Animals fasted: Yes, overnight with a maximum of 24 hours before blood sampling, but water was available.
- How many animals: 10 selected animals/sex/group
Blood samples at a target volume of 1.0 mL were collected into tubes without anticoagulant. Blood samples were processed for serum and used for measurement of both T4 and TSH. Any remaining sample was discarded.

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):

For all surviving F0-males and Cohort 1A F1-males, the following assessments were performed:
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. Evaluation was performed for all samples.
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.
As for one Group 4 F0-male, abnormalities were noted in the left epididymis, the left side organ(s) was fixed in modified Davidson's solution, and the right side organ was used for evaluation of sperm numbers.

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 until weaning:
- Mortality/morbundity: twice daily until weaning. The number of live and dead pups was determined on PND 1 and daily thereafter.
- Clinical observations: at least once daily
- Body weights: Live pups were weighed individually on PND 1, 4, 6, 7, 8, 9, 11, 13, 15, 17, 19 and 21. Several litters were also weighed on PND 8.
- Sex: externally determined for all pups on PND 1, 4 and 13.
- Anogenital distance (AGD): 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 on PND 13.

The following parameters were examined in F1 offspring from weaning onwards (Cohorts 1A, 1B, 2A and 3):
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- General health/mortality, simultaneously with the mortality/moribundity check of the dam

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were conducted in a standard arena at weekly intervals during the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed weekly from weaning onwards. 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, 1B, and 2A, a terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was quantitatively measured weekly, from weaning onwards up to the day prior to scheduled necropsy.

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

VAGINAL PATENCY: Yes
- Time schedule for examinations: monitored daily for alle females from PND 25 onwards until vaginal patency was present, by visual inspection of the vaginal area.

BALANOPREPUTIAL SEPARATION
- Time schedule for examinations: monitored daily for all males from PND 35 onwards until balanopreputial separation was present, by visual inspection of the genital area.

STAGE OF ESTROUS CYCLE
Estrous stages were determined by examining the cytology of a vaginal lavage sample, taken on the day of scheduled necropsy.

The following parameters were examined in cohort 1A animals:
ESTOUS CYCLE ETERMINATION
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.

HAEMATOLOGY
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight with a maximum of 24 hours before blood sampling, but water was available.
- How many animals: 10 selected animals/sex/group
- Parameters checked: according to OECD TG 443

CLINICAL CHEMISTRY
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. on the day of scheduled necropsy
- Animals fasted: Yes, overnight with a maximum of 24 hours before blood sampling, but water was available.
- How many animals: 10 selected animals/sex/group
- Parameters checked: according to OECD TG 443

URINALYSIS
- Time schedule for collection of urine: Overnight (15-20 hrs)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: according to OECD TG 443

THYROID HORMONE
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. on the day of scheduled necropsy (cohort 1A and 2B), at culling
- Animals fasted: Yes, overnight with a maximum of 24 hours before blood sampling, but water was available (cohort 1A only).
- How many animals: 10 selected animals/sex/group (cohort 1A and 2B), on PND 4 from two surplus pups per litter
Blood samples at a target volume of 1.0 mL (Cohort 1A), 0.5 mL (pooled PND 4 pups) and 0.4 mL (Cohort 2B PND 21-22 pups) were collected into tubes without anticoagulant. Blood samples were processed for serum.
Serum of Cohort 1A animals and Cohort 2B animals (i.e. PND 21-22 pups) was used for measurement of both T4 and TSH. Pooled serum of culled PND 4 pups was used for measurement of T4. Any remaining sample was discarded.


ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY (cohort 2A):
- Acoustic startle response:
Acoustic startle response (habituation) was assessed using the StartleMonitor System (Kinder Scientific, Poway, USA). This was performed once between PND 23-25 in a sound-attenuated room.
To the extent possible, treatment groups were balanced across devices and the time of testing was counterbalanced across dose group and sex. The animals were tested in sets of up to 3. The test sessions consisted of a five-minute acclimation period with a 65 ± 5-dB broadband background white noise. The startle stimulus for each trial was a 115 ± 5-dB mixed frequency noise burst stimulus (approximately 20 milliseconds in duration). Responses were recorded during the first 250 milliseconds following onset of the startle stimulus for each trial. The test session consisted of 50 trials with an eight-second intertrial interval. Average response amplitude (AveN), average maximum response amplitude (MaxN) and average latency to achieving the maximum response amplitude (Tmax) was analyzed in five blocks of 10 trials each.

- Functional observation battery: tests were conducted once between PND 63-75 in the order of sequence indicated below and were divided between several days.
1. Detailed clinical observations, consisting of a number of tests conducted in- and out-side the home cage
2. Rectal temperature, measured immediately after the detailed clinical observations.
3. Locomotor activity, tested using the Kinder Scientific Motor Monitor System. Recording period was one hour under normal laboratory light conditions.
To the extent possible, treatment groups were balanced across devices and the time of testing was counterbalanced across dose groups. Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.
4. Hearing ability.
5. Fore- and hindlimb grip strength, recorded per animal as the mean of three measurements, using a grip strength meter (Series M4-10, Mark-10 Corporation).
6. Pupillary reflex .
7. Landing (hind) foot splay, recorded per animal as the mean of three measurements.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY (cohort 3 and positive control animals):
All F1-animals of Cohort 3 and all positive control animals were immunized once, 5 days before scheduled necropsy (i.e. once between PND 48-54) via intravenous injection into the tail vein (approximately 1 mL/min) with 1.0 mL of the 300 µg/ml of Keyhole Limpet Hemocyanin KLH solution.
For the positive control animals, this immunization was performed 2 to 4 hours after treatment with cyclophosphamide.
For Cohort 3 animals, this immunization was performed at least 2 hours after treatment with the test item formulations/vehicle.
The animals were restrained during the injection procedure and injected using a disposable needle and butterfly needle. After injection, the injection site was marked with indelible ink.
On the day of KLH injection, clinical sign observations was performed after this injection for animals of Cohort 3 and the positive control animals.

Inject site evaluation: at least daiy, starting one day after KLH injection up to and including the day before scheduled necropsy
The irritation scores and a description of all other (local) effects were recorded as part of the “1-hour post-dose” observation. Adjacent areas of skin of each animal served as controls.
The irritation was assessed according to the following numerical scoring system. At each observation, the highest scores given were recorded:

Erythema and eschar formation:
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) 4
Where signs of necrosis or corrosion (injuries in depth) prevent erythema scoring, the maximum grade for erythema (= 4) is given.

Oedema formation:
Minimal oedema 1
Slight oedema 2
Moderate oedema 3
Severe oedema 4

In-life procedures positive control animals:
- Mortality/moribundity checks: twice daily
- Clinical observations: at least once daily, up to the day prior to necropsy.
- Body weight: Animals were individually weighed once, on the first day of cyclophosphamide treatment.
- Food and water consumption was not determined for positive control animals.

T-Cell Dependent Antibody Response (TDAR) Assay (cohort 3 and positive control animals)
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. pre-immunisation and 5 day after immunization (PND 53-59)
- Animals fasted: No
- How many animals: 10 selected animals/sex/group (cohort 3) and 10 selected animals/sex (positive control animals)
Blood samples at a target volume of 0.3 mL were collected into tubes without anticoagulant. Blood samples were allowed to clot for at least 30 minutes and centrifuged within 2 hours after collection. Within 1 hour after centrifugation, serum of these samples were divided into 2 aliquots and subsequently stored in labeled polypropylene tubes at =-75°C until analysis.
The antibody response to the immunization with KLH was determined by measuring the anti-KLH IgM levels in serum using Rat Anti-KLH IgM ELISA Kits (Life Diagnostics, Inc., West Chester, PA, USA) and an EL808™ Absorbance Microplate reader including Gen5 Secure software version 1.11.5 (BioTek Instruments, Inc., Winooski Vermont, USA) according to a validated method.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after successful mating or at the end of the mating period
- Maternal animals: All surviving animals on LD 23-25, or after total litter loss, or failure to deliver

GROSS NECROPSY
- 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.

Organ Weights and Tissue Collection/Preservation – F0-Generation
Organs were weighed at necropsy for all scheduled euthanasia animals according to the guideline. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. 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 (using the terminal body weight) were calculated.
Representative samples of the tissues were collected from all animals, preserved, processed and evaluated according to the guideline.
For females which failed to deliver a complete litter, uterine contents (i.e. any fetuses, placenta and implantation sites) was fixed (if applicable), but was not be examined histopathologically in first instance.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at:
Cohort 1A: PND 87-97
Cohort 1B: PND 97-110
Cohort 2A: PBD 76-90
Cohort 2B: PND 21-22
Cohort 3: PND 53-59
Positive control animals: PND 53-59

Unscheduled Deaths– F1 -Generation
Pups sacrificed in extremis on or after PND 7 were euthanized by an intraperitoneal injection of sodium pentobarbital.
Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally) and externally examined with emphasis on developmental morphology. For pups found dead or sacrificed in extremis from PND 14 onwards a limited necropsy was performed including sex determination (both externally and internally).
Descriptions of all external abnormalities were recorded. 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. Sex was determined both externally and internally. Pups were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities were recorded.

Spare F1-animals which were not assigned to one of the Cohorts were sacrificed between PND 22-24 by intraperitoneal injection of sodium pentobarbital. Animals were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development, and sex was determined (both externally and internally). Descriptions of all external abnormalities were recorded.

Cohort 1A
Cohort 1A animals surviving to scheduled necropsy were deprived of food overnight (with a maximum of 24 hours) before necropsy, weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Organs were weighed and tissues prepared according to the guideline.

Sperm Analysis – Cohort 1A
For all males of Cohort 1A, the following assessments were performed: 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. Evaluation was performed for all samples.
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.

Splenic Lymphocyte Subpopulation Analysis – Cohort 1A
From 10 selected animals/sex/group of Cohort 1A, splenic lymphocyte subpopulation analysis was performed at termination. 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:

Parameter (Abbreviation) Unit Markers for identification
T-cells % Lymphoid cells CD3+/CD45RA-
T-helper cells % Lymphoid cells CD3+/CD4+/CD8-
T-cytotoxic cells % Lymphoid cells CD3+/CD4-/CD8+
B-cells % Lymphoid cells CD3-/CD45RA+
NK-cells % Lymphoid cells CD3-/CD161a+
Ratio T-helper cells/ T-cytotoxic cells (Th/Tc) - -

Cohort 1B
Cohort 1B animals were not deprived of food overnight before necropsy. These animals were weighed weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

Cohort 2A and 2B
The animals were not deprived of food overnight before necropsy and a terminal body weight was recorded. The animals were first anaesthetized using isoflurane and subsequently sacrificed by whole body (in situ) perfusion using heparinized saline (0.9% NaCl) followed by a 4% paraformaldehyde solution (adjusted to pH 7.4; HCl, KCl, NaH2PO4 x H2O, Na2HPO4 x 2H2O, paraformaldehyde and NaOH, aqua dest.).
All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.
After perfusion, the cranium was removed, exposing the brain. The skull including the brain was placed in 10% buffered formalin and allowed to fix for at least 7 days prior to removal from the skull.
The fixed brains were removed and weighed, and the length and maximum width of the brain was measured for all animals selected for neuropathology. Subsequently, the brain was fixed in 10% buffered formalin together with selected PNS tissues.
Morphometric (quantitative) analyses of CNS tissues was performed for Cohort 2A and 2B animals of Groups 1 and 4.

Cohort 3 and Positive Control Animals
Scheduled necropsy of Cohort 3 was conducted on PND 53-59 (except for one animal which was necropsied on PND 49). Positive control animals were euthanized on the same date(s). The animals were deeply anaesthetized using isoflurane and subsequently exsanguinatedand were subjected to a limited examination, with special attention being paid to the reproductive organs.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

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).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means was applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
The startle data set and the follicle count data set (at least 3 groups) was compared using an overall Kruskal-Wallis. Whenever, the overall test is significant, the Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group.

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:

Mating index males (%): Number of males mated/Number of males paired x 100
Mating index females (%): Number of females mated/Number of females paired x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index males(%): Number of pregnant females/Number of males mated x 100
Fertility index females(%): Number of pregnant females/Number of females mated x 100
Gestation index (%): Number of females with living pups on Day 1/Number of pregnant females x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Post-implantation survival index (%): Total number of offspring born/Total number of uterine implantation sites x 100
Live birth index (%): Number of live offspring on Day 1 after littering/Total number of offspring born x 100
Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
Viability index (%): Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering x 100
Weaning index (%): Number of live offspring on Day 21 after littering/Number live offspring on Day 4 (after culling) x 100
Percentage live males at weaning (%): Number of live male pups on Day 21 after littering/Number of live pups on Day 21 after littering x 100
Percentage live females at weaning (%): Number of live female pups on Day 21 after littering/Number of live pups on Day 21 after littering x 100

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were noted during daily detailed clinical observations that were considered to be signs of toxicity. No findings were noted during the weekly arena observations in this study.
Salivation seen after dosing among all test item treated groups in a dose-related manner was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. shortly after dosing). This sign may be related to taste of the test substance.
Other 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 considered signs of no toxicological relevance.

Mortality:

no mortality observed

Description (incidence):

No mortality related to treatment with the test item occurred during the study period.
One female of the 200 mg/kg/day group was euthanized on Lactation Day 3, as she had a total litter loss.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg bw/day, mean body weight gain of females was statistically significantly lower than controls on Days 14 and 21 of the lactation phase (0.7x and 0.5x, respectively), which was considered to be related to treatment with the test item. Mean body weight gain at the end of the lactation phase was 0.5x of the control mean. Two females at 200 mg/kg/day showed weight loss of 8-11% and 1-5% of body weight on PND 1 throughout the lactation phase, respectively.
Other variations in mean absolute body weights noted during the premating and post-coitum phase were considered not to be related to treatment. During the premating period these variations consisted of slightly higher mean absolute body weights in females at 40, 100 and 200 mg/kg/day. A level of statistical significance was achieved on most occasions during the premating phase at 100 and 200 mg/kg/day, and at 40 mg/kg/day on Day 22 only. Mean body weight gain at the end of the premating period was similar between the groups.
During the post-coitum phase, these variations consisted of statistically significantly higher mean absolute body weights at 200 mg/kg/day on Day 7. Mean body weight gain during the post-coitum phase was similar between the groups. Since a clear dose-related trend was absent and since these variations were of a slight degree and/or not consistently noted as treatment progressed, they were considered not to be related to treatment.
Body weights and body weight gain of males remained in the same range as controls over the treatment period.

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 was considered not affected by treatment with the test item in males and females.
A statistically significant higher mean absolute food consumption was recorded for females at 200 mg/kg/day over Days 1-15 of the premating phase. However, since this apparent difference was very minor and did not become more pronounced as treatment progressed, this was considered not to be related to treatment.

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):

Haematological parameters of treated rats were considered not to have been affected by treatment with the test item.
Any statistically significant changes in haematology 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.

Coagulation
At 200 mg/kg bw/day, a statistically significantly elongated prothrombin time (PT) was recorded for males (1.08x; mean remained within the internal control range (mean = 17.6; P5 – P95 = 15.50-19.40 (n=79)).
At 40 and 100 mg/kg bw/day in males and females, and at 200 mg/kg bw/day in females, coagulation parameters were considered not to have been affected by treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Higher mean cholesterol was recorded in males at 200 mg/kg bw/day (1.26x; mean remained within the internal control range (mean = 1.81; P5 – P95 = 1.315-2.465 (n=80)), which was considered to be related to treatment.
Slightly lower total protein in males at 100 and 200 mg/kg bw/day and slightly lower albumin in males at 40, 100 and 200 mg/kg/day were considered not to be related to treatment with the test item, since these variations occurred in the absence of a dose-related trend and since means remained within the internal control range.
Clinical chemistry parameters in F0-females were considered not affected by treatment.
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.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis revealed no changes that were considered to be related to treatment with the test item.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

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 observations.
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 findings: neoplastic:

not examined

Other effects:

not examined

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 with the test item.
Most females had regular cycles of 4 days (one female at 100 mg/kg bw/day had a cycle length of 5 days). For two control females, regularity of the estrous cycle could not be determined. An irregular cycle was noted for one female at 40 mg/kg/day (with normal litter). Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Sperm motility, concentration and morphology were considered not affected by treatment with the test item.
A statistically significantly higher or lower mean epididymal sperm count was recorded at 40 and 100 mg/kg bw/day, respectively. At 40 mg/kg bw/day, the standard deviation from the mean was relatively high, and 6/25 individual values at this dose level exceeded the historical data range. However, these variations in mean epididymal sperm count occurred in the absence of a dose-related trend, and in absence of macroscopic changes on male reproductive organs and means remained within the internal control range. Therefore, these variations were considered not to be related to treatment.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating index was considered not affected by treatment with the test item.
The mating indices were 100% for the control group, and 40 and 100 mg/kg bw/day groups. Two females at 200 mg/kg bw/day did not show evidence of mating, resulting in a mating index of 92% in this group which was considered to be within normal ranges (mean = 99 (n=7 studies; no confidence interval due to the limited number of data); Historical control data for OECD422 studies (mean: 99; P5 – P95 = 90-100 (n=120 studies)). As such, the incidence of two non-mated females in this group was considered not to be related to treatment, also given the absence of treatment-related changes in other reproductive parameters.

Precoital time was considered not affected by treatment with the test item. Mean precoital time was 2.8 days in the control group, 3.8 days at 40 and 100 mg/kg bw/day and 3.3 days at 200 mg/kg bw/day.
Most females showed evidence of mating within 5 days, except for three females at 40 mg/kg bw/day, two females at 100 mg/kg bw/day and two females at 200 mg/kg bw/day for which mating took 13 or 14 days. As this variation in precoital time remained within the normal range of biological variation, occurred at low incidence and in absence of a dose-related trend, this was considered to be unrelated to treatment.

Number of implantation sites was considered not affected by treatment with the test item.

Fertility index was considered not affected by treatment. The fertility indices were 100% for the control and 200 mg/kg/day group, and 88% for the 40 and 100 mg/kg/day groups.
A total of three females at 40 mg/kg/day and three females at 100 mg/kg/day were not pregnant. In the absence of a dose-related incidence of non-pregnancy and since mean fertility indices remained within the internal control data range , this was considered not to be related to treatment.

Gestation index and duration of gestation were considered not affected by treatment with the test item; the gestation indices were 100% for all dose groups. All pregnant females had live offspring. Mean duration of gestation ranged from 21.3 to 21.5 days across the dose groups.

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.
For two females at 100 mg/kg bw/day and one female at 200 mg/kg bw/day, the number of pups was higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation. No toxicological relevance was attached to this finding in the current study.

The total number of offspring born compared to the total number of uterine implantations was considered not affected by treatment with the test item.
Post-implantation survival index was 94% for the control group and 95, 100 and 97% for the 40, 100 and 200 mg/kg bw/day groups, respectively. These means remained within the range considered normal for rats of this age and strain (due to the limited number of data, internal control data for OECD 421/422 studies were used (period 2015-2018)): mean: 92%, P5-95: 83-99 (n=98 studies)).
Litter size was considered not affected by treatment with the test item. Live litter sizes were 11.2, 11.2, 11.5 and 11.3 living fetuses/litter for the control, 40, 100 and 200 mg/kg bw/day groups, respectively.

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity F0

Effect level:

>= 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse effects up to and including the highest dose level.

Remarks on result:

other: Possible adversity of the higher TSH in males at 40, 100 and 200 mg/kg/day and higher total T4 in males at 100 and 200 mg/kg/day could not be assessed within this type of study and was therefore not taken into account when determining the NOAEL.

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity F0

Effect level:

>= 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of adverse effcets on reproduction parameters up to and including the highest dose level.

Key result

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to represent signs of toxicity.
Primary clinical signs among pups that were found dead or were sacrificed in extremis before weaning consisted of abdominal swelling and pallor (PND 10) for one control pup, lean appearance (PND 12 and 13) and lateral recumbency (PND 13) for one pup at 40 mg/kg bw/day, and dehydrated and lean appearance (PND 12) for one pup at 100 mg/kg bw/day. Given that the incidence of these findings showed no relationship to the dose and since their unscheduled sacrifice was considered to be gavage-related, these clinical signs were considered not to represent signs of toxicity.
The nature and incidence of other clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Breathing rales were recorded for 3/60 males at 40 mg/kg bw/day, 12/60 males and 12/60 females at 100 mg/kg bw/day, and 17/60 males and 8/60 females at 200 mg/kg bw/day, primarily during the first two weeks of treatment post-weaning. Rales were also recorded for 2/60 control females.
No other treatment-related clinical signs were noted among surviving animals during daily detailed clinical observations or during weekly arena observations.
Salivation was recorded after dosing among several males and females at 100 mg/kg bw/day and most males and females at 200 mg/kg w/day during the treatment period with a dose-related incidence. This clinical sign was considered to be a physiological response related to gavage administration of the test item rather than a sign of systemic toxicity, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
No clinical signs were noted during treatment of Positive Control animals.
Clinical signs of animal that did not survive until scheduled necropsy are specified under Mortality - F1 generation.
Any other clinical signs noted during the treatment period among surviving animals 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 / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment with the test item. One pup at 100 mg/kg bw/day and one pup at 200 mg/kg/day were found dead at first litter check. 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.

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 with the test item. Viability indices were 100% for the control group and 40 and 100 mg/kg bw/day groups, and 98% for the 200 mg/kg bw/day group. One pup at 40 mg/kg bw/day, one pup at 100 mg/kg bw/day and five pups at 200 mg/kg bwday were found missing between PND 2 and 4. These pups were most likely cannibalised. No toxicological relevance was attributed to these 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.

The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was considered not affected by treatment with the test item.
The weaning indices were 97% for the control group, and 90, 93 and 86% for the 40, 100 and 200 mg/kg bw/day groups, respectively. Note that the weaning index did not include dead animals that were replaced by another animal.
Five pups of the control group, seventeen pups at 40 mg/kg bw/day, twelve pups at 100 mg/kg bw/day, and twenty-nine pups at 200 mg/kg bw/day did not survive the scheduled treatment period. This included one male at 40 mg/kg bw/day and four males at 200 mg/kg bw/day that were sacrificed in extremis or found dead between PND 21 and 25, i.e. after weaning. Most of these pups were sacrificed in extremis or found dead between PND 10 and 20. Three pups at 40 mg/kg bw/day and one pup at 200 mg/kg bw/day were found missing between PND 14 and 22 and were most likely cannibalised.
Most of these mortality cases occurred on a single day, when a total number of 1, 3, 9 and 21 pups were found dead or missing at 0, 40, 100 and 200 mg/kg bw/day, respectively. The number of pups that died outside this date in the period between culling and weaning was 4, 14, 3 and 8 at 0, 40, 100 and 200 mg/kg bw/day, respectively. This incidence did not show a correlation to the dose administered. The most relevant macroscopic observations for these pups that did not survive until scheduled necropsy were essentially confined to lungs that were not collapsed, and fluid was found in the thoracic and/or abdominal cavity. None of these macroscopic findings were recorded for F1 animals that survived until scheduled necropsy. The nature of these findings is suggestive of a gavage-related occurrence.
Overall, it was considered that this pup mortality was not related to toxicity of the test item but had occurred principally as a result of the dosing procedure. Any clear relationship between the dose and mortality incidence was absent, most cases of mortality occurred on a single day, and the nature of the macroscopic observations was specific for the pups that were found dead/sacrificed and were suggestive of a gavage-related occurrence.

No post-weaning mortality occurred that was considered to be related to treatment with the test item.
A total of five animals were sacrificed in extremis or found dead after weaning between PND 21 and 25. Except for one female, all these animals could be replaced by another animal in order to supplement the cohorts. At 40 mg/kg bw/day, one Cohort 1B male was found dead on PND 23. This animal showed rales and gasping prior to death and a necropsy finding (lungs not collapsed) similar to that seen for several pups that were sacrificed/found dead before weaning. At 200 mg/kg bw/day, four males were sacrificed in extremis or found dead after weaning between PND 21 and 25, i.e. two Cohort 1B animals, one Cohort 2B animal and one Cohort 3 animal. Three of these males at 200 mg/kg bw/day showed hunched posture, uncoordinated movements, piloerection, deep respiration, rales, brown staining of the genital region, and/or lethargy prior to sacrifice.

No further mortality occurred on subsequent treatment days among any group. It was therefore conceivable that the death/sacrifice of these animals after weaning had a similar cause as those pups that died or were found dead prior to weaning. In the absence of an overall dose-related trend in mortality incidence (pre- and post-weaning deaths combined), these deaths were considered not 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 affected by treatment with the test item. Means of test item treated groups were similar to the control group means on all occasions.
No changes in body weight or body weight gain of F1 animals post-weaning were noted that were considered to be related to treatment with the test item.
In females, statistically significant increases in body weight gain in Week 29 and 36 (less than 10% different to control means) were considered not related to treatment as a dose-related trend was absent and these increases were transient (final body weights were similar between groups).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption (before and/or after correction for body weight) was considered not affected by treatment with the test item.

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):

The following statistically significant changes were noted in haematology parameters that were considered to be related to treatment with the test item.
- Lower platelet counts in males at 200 mg/kg bw/day (0.87x; mean remained within the internal control range (mean = 726; P5 – P95 = 601-851 (n=75)))
- Higher red cell distribution width (RDW) in females at 200 mg/kg bw/day (1.06x; mean remained within the internal control range (mean = 11.0; P5 – P95 = 10.10-12.40 (n=75)))
Any other statistically significant changes in haematology 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.

Coagulation parameters were considered not affected by treatment with the test item. The statistically significantly lower activated partial thromboplastin time (APTT) in males at 100 mg/kg bw/day occurred in the absence of a dose-related trend. Also, the opposite effect (i.e. a delayed APTT) is expected in case of target organ toxicity. Therefore, this variation was considered not to be related to treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes were noted in clinical chemistry parameters that were considered to be related to treatment with the test item.
- Higher urea in males at 200 mg/kg bw/day (1.15x; mean remained within the internal control range (mean = 4.9; P5 – P95 = 3.75-6.40 (n=80)))
- Higher cholesterol in males and females at 100 and 200 mg/kg bw/day (1.29x and 1.25x, respectively for males, and 1.39x and 1.42x, respectively for females; means remained within the internal control range (males: mean = 1.90; P5 – P95 = 1.295-2.530 (n=80);females: mean = 1.40; P5 – P95 = 0.825-2.175 (n=80)))
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 (including higher urea and chloride in females at 40 and 200 mg/kg bw/day, and higher alkaline phosphatase activity (ALP) in females at 200 mg/kg bw/day), or were considered not toxicologically relevant as the opposite effect would be expected in case of target organ toxicity (lower alanine aminotransferase activity (ALAT) in males at 200 mg/kg bw/day). As such, these differences were considered not related to treatment.

Serum T4 levels in male and female pups culled at PND 4, and serum T4 and TSH levels in male and female pups of Cohort 2B at PND 21-22 were considered not affected by treatment with the test item. Mean total T4 values of Cohort 2B males showed an apparent downward trend over the dose groups, but the variation in means across the dose groups was similar to that recorded for Cohort 2B females where such an apparent trend was not visible.
A higher T4 was recorded at 100 and 200 mg/kg bw/day in Cohort 1A males and females at PND 89-92 (1.21x and 1.33x, respectively for males, and 1.41x and 1.58x, respectively for females (not statistically significant for females at 100 mg/kg bw/day)), which was considered to be related to treatment with the test item. However, means were within the internal control range (males: mean = 6.23; P5 – P95 = 4.180-8.220 (n=70); females: mean = 3.54; P5 – P95 = 1.810-6.060 (n=70)).
TSH was considered not affected by treatment with the test item.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis parameters were considered not affected by treatment with the test item. Any statistically significant changes in urinary parameters occurred in the absence of a dose-related response and were therefore considered not related to treatment.

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

Balanopreputial separation (prepuce opening) in males and vaginal patency (vaginal opening), occurrence of first estrus and time between vaginal opening and first estrus in females was considered not affected by treatment with the test item.
At 200 mg/kg bw/day, a statistically significantly longer period to achieving vaginal opening was recorded (30.5 days vs. 29.1 days in the control); the mean remained well within the internal control data range, as did means of all other groups. Also, a clear dose-related trend was absent, and the control mean was at the lower margin of the internal control data range. The vast majority of individual values remained within the concurrent control and/or internal control data range: 3/60 values (34, 34 and 38) were slightly outside the concurrent control range (control range: 25-33; at 200 mg/kg/day), and 1/60 values (38) were outside the internal control data range. Mean body weight on the day of vaginal opening was considered not affected by treatment. Therefore, overall, these variations in vaginal opening were considered not related to treatment with the test item.
At 200 mg/kg bw/day, a statistically significantly longer period to achieving first estrous was recorded (34.6 days vs. 32.5 days in the control). The control mean was at the lower margin of the internal control data range. Most individual values remained within the concurrent control data range: 2/20 values (38 and 37) were slightly outside the concurrent control range (control range: 30-36; at 200 mg/kg bw/day), and all individual values remained within the internal control data range. The means of all groups remained well within the internal control data range. Therefore, overall, these variations in the period to achieving first estrous were considered not related to treatment with the test item.
The interval between day of vaginal opening and day of first estrous was not significantly different between the groups. All individual values remained within the concurrent control range and internal control data range. The means of all groups remained well within the internal control data range.

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 affected by treatment with the test item.
Mean corrected anogenital distance was 1.46, 1.49, 1.45 and 1.44 mm in males, and 0.57, 0.56, 0.54 and 0.56 mm in females for the control, 40, 100 and 200 mg/kg bw/day groups, respectively.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Areola/nipple retention was considered not affected by treatment. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Organ weights of Cohort 1A and 1B animals were considered not affected by treatment.
In Cohort 1A males treated at 200 mg/kg bw/day, a statistically significantly higher and possible test item-related increase in thyroid gland weight (absolute (+18%) and relative to body weight (+15%)) and lower adrenal gland weight (absolute (-11%) and relative to body weight (-12%)) was recorded. Similar organ weight variations were not recorded for Cohort 1B males which were necropsied in a similar time interval as Cohort 1A animals. Also, means of these organ weights in Cohort 1A were similar to those recorded across Cohort 1B groups, and remained within the internal control data range (see below). Moreover, these organ weight changes occurred without any macroscopic or microscopic correlate. Therefore, these statistically significant changes in thyroid and adrenal gland weights in Cohort 1A animals were considered not to be of toxicological relevance.
Adrenal and thyroid gland weights were considered not affected in females.
The statistically significantly lower seminal vesicle to body weight ratio recorded for Cohort 1A males at 200 mg/kg bw/day was considered not to be related to treatment, since mean seminal vesicle to body weight ratios of Cohort 1B males which were necropsied in a similar time interval as Cohort 1A animals were similar to the control mean. Also, the difference to the control mean in Cohort 1A was slight, absolute mean seminal vesicle weight in Cohort 1A at 200 mg/kg bw/day was similar to the control mean and remained within the internal control range (see below). Moreover, this variation in seminal vesicle to body weight ratio occurred without any macroscopic or microscopic correlate.
Any other differences, including those that reached statistical significance, were considered to be incidental and not related to the test item.

Internal control data
Thyroid gland weight - absolute (gram): mean = 0.016; P5 – P95 = 0.0105-0.0231 (males, Cohort 1A; n=160).
Thyroid gland weight - relative (% from terminal body weight): mean = 0.005; P5 – P95 = 0.0031-0.0063 (males, Cohort 1A; n=159).
Adrenal gland weight - absolute (gram): mean = 0.060; P5 – P95 = 0.0460-0.0755 (males, Cohort 1A; n=160).
Adrenal gland weight - relative (% from terminal body weight): mean = 0.017; P5 – P95 = 0.0130-0.0221 (males, Cohort 1A; n=159).
Seminal vesicle weight – relative (% from terminal body weight): mean = 0.302; P5 – P95 = 0.2052-0.4189 (males, Cohort 1A; n=159).

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 with the test item.
There were no test item-related gross observations in Cohort 2B (PND 21-22), Cohort 3 (PND 53-59), Cohort 2A (PND 76-100), Cohort 1A (PND 89-95) and Cohort 1B (= PND 90).
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings:

no effects observed

Description (incidence and severity):

Cohort 1A
There were no test item-related microscopic observations.
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 200 mg/kg bw/day group females (Cohort 1A) when compared to control group females. Any variation between group mean counts represented biological variability and were not statistically significant.

Neuropathology and Morphometry (Cohorts 2A and 2B)
Absolute and relative fixed brain weights at PND 21-22 (Cohort 2B) and PND 76-90 (Cohort 2A) were considered not affected by treatment with the test item.
Brain dimensions (length and width of brain) at PND 21-22 (Cohort 2B) and PND 76-90 (Cohort 2A) were considered not affected by treatment with the test item. The statistically significantly lower brain width of Cohort 2B males at 100 mg/kg/day occurred in the absence of a dose-related trend and was therefore considered not related to treatment.
There were no changes in morphometry of the brain of Cohort 2B animals (PND 21-22) and Cohort 2A animals (PND 76-90) that were considered to be related to treatment. At PND 21-22, Cohort 2B females in the 200 mg/kg bw/day dose group had a significantly lower mean height of the cerebellum compared to controls. This finding was no longer present at PND 76-90 and although not statistically significant, mean cerebellar height of the 200 mg/kg bw/day Cohort 2A females was greater than controls.
The Cohort 2A, 200 mg/kg bw/day males had a statistically significantly greater mean height of the cerebellum compared to controls at PND 76-90. Given the inconsistent direction of change in the cerebellar height in the 200 mg/kg bw/day females at PND 21-22 and PND 76-90, these findings are considered unrelated to DGEBADA administration. Additionally, the increased cerebellar height in the 200 mg/kg bw/day males on PND 76-90 is in a direction opposite of what is expected for a toxicologically relevant effect.

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio F1 pups
Sex ratio was considered not affected by treatment.

Estrous Cycle (Cohort 1A)
Length and regularity of the estrous cycle were not affected by treatment. For most females, regular cycles of 4 to 5 days were recorded between PND 75 and 88. One female at 40 mg/kg bw/day and one female at 200 mg/kg bw/day were acyclic. Given the incidental nature and the absence of a dose-related incidence, these single occurrences of an acyclic estrous cycle were considered not to indicate a relation to treatment with the test item.

Sperm Analysis (Cohort 1A)
Sperm motility, concentration and morphology were considered not affected by treatment with the test item.

Splenic Lymphocyte Subpopulation (Cohort 1A)
Splenic lymphocyte subpopulations (Cohort 1A) were considered not affected by treatment with the test item. No statistically significant changes were observed in splenic lymphocyte subpopulation parameters.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Acoustic Startle Response:
At 200 mg/kg/day, males showed a statistically significantly higher average response amplitude (both as overall mean response (1.38x), and during the first four test blocks (1.64x, 1.38x, 1.42x and 1.42x for blocks 1, 2, 3 and 4, respectively)), maximum response amplitude (both as overall mean (1.23x) and during the first test block (1.47x)) and latency to achieving the maximum response amplitude (both as overall mean (1.27x) and during the first test block (1.46x)). These changes were considered to be related to treatment with the test item.
At 40 and 100 mg/kg/day (males and females), and 200 mg/kg/day (females), acoustic startle response (both as overall mean and per block) was considered not affected by treatment. The statistically significantly lower average and maximum response amplitude at 100 mg/kg/day in females occurred in the absence of a dose-related trend, and was therefore considered not to be related to treatment with the test item.

Detailed clinical observations:
Detailed clinical observations revealed no symptoms that were considered to be related to treatment with the test item.
The clinical symptoms that were observed were considered to be within the normal range of behavioural findings for this type of study, and were generally also observed in control animals. These findings were therefore considered not to be related to treatment.
Decreased locomotor activity was recorded for 2/10 and 1/10 males at 100 and 200 mg/kg/day, respectively. Increased urinating was recorded for 1/10 females at 200 mg/kg/day.
Whilst these symptoms were not recorded among control animals, they were considered not to be related to treatment with the test item given their low incidence, and absence of a clear dose-related response.

Rectal temperature:
Rectal temperature was considered not affected by treatment with the test item.

Motor activity :
Motor activity was considered not affected by treatment with the test item. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Slightly higher mean activity levels (ambulations and total movements) were recorded for females at 40 and 100 mg/kg/day, but these did not achieve a level of statistical significance and occurred in the absence of a dose-related trend. As such, these variations were considered not to be related to treatment with the test item.

Functional observations :
Functional observation parameters (hearing ability, pupillary reflex, foot splay and grip strength) were considered not affected by treatment with the test item. Hearing ability and pupillary reflex were normal in all examined animals. Mean foot splay values of males at 200 mg/kg/day appeared lower than controls but without statistical significance.
A statistically significantly higher forelimb grip strength was recorded for females at 100 mg/kg/day. In absence of a dose-related response, this variation was considered to be unrelated to treatment with the test item.

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

All animals were negative for the presence of anti-KLH IgM antibodies before the administration of KLH.
The incidence of non-responders (< LLOQ) to KLH (IgM) in the control group was higher than expected for this assessment. Indeed, in the vehicle control group, there were 5/10 males and 2/10 females with non-detectable levels of anti-KLH IgM antibodies. It is unclear why a high incidence of vehicle control animals mounted a weak response to KLH in the present study. This trend was also observed in most dosed groups. There were at least 3 animals per sex/group with detectable responses to KLH (IgM), wBased on the data available from the responding animals, there were no DGEBADA related effects on the levels of anti-KLH IgM antibodies detected at any dose levels.
Based on the data available from the responding animals, there were no DGEBADA related effects on the levels of anti-KLH IgM antibodies detected at any dose levels.
It should also be noted that no pathology findings were detected in the lymphoid organs (i.e. histopathology and organ weight) and no treatment related changes in splenic lymphocyte subpopulations were observed in this study (refer to relevant section in the Main Study Report). Therefore, overall, the data indicates that DGEBADA does not appear to induce any immunotoxic effect in young Wistar rats.

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Generation:

F1

Effect level:

>= 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of adverse effects up to and including the highest dose level.

Dose descriptor:

NOEL

Remarks:

General toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The presence of rales and higher cholesterol at 100 and 200 mg/kg bw/day in F1 males and females.

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Dose Formulation Analyses

Accuracy

In the Group 1 formulations (F0-Animals and F1-Animals), no test item was detected.

The concentrations analyzed in the formulations of Groups 2, 3 and 4 for F0-Animals, prepared for use in Weeks 1, 2, 3, 5, 13, 15, 21 and 25 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). For the formulations of Group 2, 3 and 4 (F0-Animals), prepared for use in Week 9, the mean accuracy was slightly below the target concentration (i.e. 89%, 84% and 89% of target). No cause for these slightly lower values could be found. Based on overall accuracy results however, it was considered that formulations were prepared accurately for the purpose of this study.

The concentrations analyzed in the formulations of Groups 2, 3 and 4 for F1-Animals, prepared in Week 15 and Group 2 for F1-Animals prepared in Week 25 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The high mean accuracy for the formulation of Group 2 (F1-Animals) prepared in Week 21 (i.e. 976% of target) was due to an incorrect preparation of the formulation (not used for dosing). 

Homogeneity

The formulations of Group 2 (F0-Animals) and Group 4 (F0-Animals) and Group 2 (F1-Animals) were homogeneous (i.e. coefficient of variation= 10%).

Stability

Analysis of Group 4 (F0-Animals) and Group 2 (F1-Animals) formulations after storage yielded a relative difference of= 10%. The formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 24 hours.

Mean Total T4 and TSH levels (and Fold Increase compared to Control Mean) in F₀and F₁animals.

Parameter	Dose level (mg/kg/day)	F0		PND 4	PND 21-22 (Cohort 2B)		PND 89 (Cohort 1A)
		M	F	M/F	M	F	M	F
Total T4 (µg/dL)   Mean(fold change)	0	6.25	4.98	1.46	4.65	4.32	6.09	3.06
	40	6.15 (n.a.)	5.57 (n.a.)	1.56 (n.a.)	4.39 (n.a.)	3.85 (n.a.)	6.39(1.05x)	3.45 (1.13x)
	100	7.47 (1.20x)	5.00 (n.a.)	1.59 (n.a.)	4.31 (n.a.)	4.07 (n.a.)	7.37* (1.21x)	4.30 (1.41x)
	200	8.04 ** (1.27x)	5.34 (n.a.)	1.58 (n.a.)	4.09 (n.a.)	4.19 (n.a.)	8.12 ** (1.33x)	4.83** (1.58x)
TSH (uIU/mL)   Mean(fold change)	0	0.111	0.193	-	0.079	0.086	0.153	0.079
	40	0.125 (1.13x)	0.507 (n.a.)	-	0.088 (n.a.)	0.079 (n.a.)	0.135 (n.a.)	0.072 (n.a.)
	100	0.153 (1.38x)	0.274 (n.a.)	-	0.093 (n.a.)	0.108 (n.a.)	0.168 (n.a.)	0.072 (n.a.)
	200	0.185 (1.67x)	0.246 (n.a.)	-	0.091 (n.a.)	0.111 (n.a.)	0.142 (n.a.)	0.092 (n.a.)

M: male; F: female.

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

n.a. Not applicable; no treatment-related effect or mean not in line with increasing or decreasing trend over the dose groups.

Executive summary:

 The objective of this study was to provide an evaluation of the pre- and postnatal effects of DGEBADA on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats. 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 DGEBADA 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. 

Furthermore, the information obtained from the developmental neurotoxicity and developmental immunotoxicity assessments characterized potential effects in those systems.

The dose levels in this study were selected to be 0, 40, 100 and 200 mg/kg/day, based on the results of previously conducted (reproductive) toxicity studies with oral exposure of DGEBADA in rats.

A Lactational Transfer Study (Test Facility Study No. 20172948) was performed prior to this studyto determine if DGEBADA could transfer to mother’s milk after maternal exposure fromDay 6 post-coitum to Day 13 of lactation, inclusive. All measured milk samples collected from dams on Day 13 of lactation revealed values of DGEBA diacrylate (the main compound present in the test item) that were below the lower limit of quantification (LLOQ). This result indicated that DGEBADA is not excreted in the mother’s milk at detectable levels, when administered up to a dose level of 200 mg/kg/day by once daily oral gavage from Day 6 post-coitum until Day 13 of lactation inclusive.

Chemical analyses of formulations were conducted on nine occasions during the study to assess accuracy, homogeneity and stability over 24 hours at room temperature.

For the F0-generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, estrous cycle determination,clinical pathology including measurement of thyroid hormones and urinalysis,gross necropsy findings, sperm analysis, organ weights and histopathologic examinations. 

For the F1-generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, vaginal patency and balanopreputial separation, day of first estrous, estrous cycle determination, functional observations including acoustic startle response, immunotoxicity assessments using TDAR assay),clinical pathology including measurement of thyroid hormones and urinalysis,gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations, neurohistopathological examinations and morphometric analysis of brain tissue.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 and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormones).

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

No adverse changes were noted in any of the F0-Generation parameters investigatedup to the highest dose level tested (200 mg/kg/day).

No mortality and no adverse clinical signs related to treatment occurred during the study period.

Non-adverse changes in body weight were recorded among females, however the lower mean body weight gain of females at 200 mg/kg/day was considered to be treatment-related. Statistically significant lower than controls on Days 14 and 21 of the lactation phase (0.7x and 0.5x respectively). The mean body weight gain at the end of the lactation phase was 0.5x of the control mean. The other variations in mean absolute body weights noted during the premating and post-coitum phases were considered not to be related to treatment.

Food consumption before or after correction for body weight was considered not affected by treatment with the test item in males and females.

Haematological parameters of treated rats were considered not to have been affected by treatment with the test item.

Non-adverse changes in coagulation and clinical chemistry parameters consisted of an elongated prothrombin time and higher cholesterol in males at 200 mg/kg/day are considered to be related to treatment. Since means of these parameters remained within the internal control range, and had no corroborative morphological lesions, these were considered non-adverse.

A trend towards higher thyroid stimulating hormone (TSH) was recorded in males at 40, 100 and 200 mg/kg/day, but means remained within the internal control range. Also, higher total T4 was recorded in males at 100 and 200 mg/kg/day; mean was outside the internal control range at 200 mg/kg/day only. Serum levels of TSH and T4 in F0-females were considered not affected by treatment. These changes in males were not accompanied by test item-related changes in thyroid morphology, but were considered as related to treatment. However, possible adversity of this effect could not be assessed within this type of study. No test item-related changes were noted in any of the other F0-Generation parameters investigated in this study (i.e. functional observations, urinalysis, macroscopic examination, organ weights and microscopic examination).

Reproduction results – F0-generation

No test item-related changes were noted in any of the reproductive parameters investigatedup to the highest dose level tested (200 mg/kg/day), i.e.sperm motility, sperm concentration and morphology, mating and fertility indices, precoital time, number of implantations, estrous cycle, 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 test item-related changes were noted in any of the developmental parameters investigated up to the highest dose level tested (200 mg/kg/day) during the gestation and lactation phases.

Five animals of the control group, seventeen animals at 40 mg/kg/day, twelve animals at 100 mg/kg/day, and twenty-nine animals at 200 mg/kg/day (including one animal at 40 mg/kg/day and four animals at 200 mg/kg/day that were sacrificed or found dead post-weaning) did not survive the scheduled treatment period. It was considered that this mortality was not related to toxicity of the test item but had occurred principally as a result of the dosing procedure. Clear relationship between the dose and mortality incidence was absent, most cases of mortality occurred on a single day, and the nature of the macroscopic observations was specific for the animals that were found dead/sacrificed and were suggestive of a gavage-related occurrence.

No test item-related changes were noted in any of the following developmental parameters investigated in this study: gestation index and duration, live birth, viability and weaning indices, parturition, sex ratio, litter size, maternal care and early postnatal pup development consisting of mortality, pups’ clinical signs, pups’ body weight, anogenital distance, areola/nipple retention, thyroid hormone levels in pups (T4 of PND 4, T4 and TSH of PND 22) and macroscopic examination of pups sacrificed at the end of the lactation period.

 

Developmental results – F1 generation (post-weaning)

A dose-related but non-adverse incidence of breathing rales was recorded at 100 and 200 mg/kg/day, primarily during the first two weeks of treatment. Incidences were 12/60 males and 12/60 females at 100 mg/kg/day, and 17/60 males and 8/60 females at 200 mg/kg/day. Incidences of rales in males at 40 mg/kg/day (3/60 males) were comparable to incidences in control females (2/60 control females), and as such these may not be attributable to the test item. Since these rales essentially resolved with continuing treatment, this was considered not an adverse finding. However, it could not be excluded that occurrence of these rales may be associated with deaths that occurred primarily prior to weaning, as most of these deaths showed lung lesions (lungs that were not collapsed, and fluid in the thoracic and/or abdominal cavity) suggestive of a gavage-related occurrence.

At 200 mg/kg/day, non-adverse treatment-related changes in acoustic startle response parameters of Cohort 2A males were recorded.These changes consisted of a higher average response amplitude (both as overall mean response (1.38x), and during the first four test blocks (1.64x, 1.38x, 1.42x and 1.42x for blocks 1, 2, 3 and 4, respectively)), maximum response amplitude (both as overall mean (1.23x) and during the first test block (1.47x)) and latency to achieving the maximum response amplitude (both as overall mean (1.27x) and during the first test block (1.46x)). Females at this dose showed no such changes in acoustic startle response parameters, and these parameters were considered not affected by treatment with the test item in males at 40 and 100 mg/kg/day. Other developmental neurotoxicity endpoints of Cohort 2A animals (detailed clinical observations, rectal temperature, hearing ability, pupillary reflex, foot splay, grip strength and motor activity) were considered not affected by treatment, and there was no apparent relationship between body weights and the level of acoustic startle response on an individual animal basis. These changes were considered not adverse since the direction of change consisted of an increased response rather than a decreased response, the auditory reflex loop was functioning at least in a comparable manner as in controls, habituation occurred as expected and recorded for control rats,means were only slightly outside historical control data range for maximum response amplitude in Block 1 and since the changes only occurred for males and not for females.

No test item-related changes were recorded for other developmental neurotoxicity endpoints determined in Cohort 2A and 2B animals, consisting of fixed brain weights, brain dimensions (length and width of brain), routine sections of brain or peripheral nervous system and morphometric analysis of the brain.

Non-adverse changes in haematology and clinical chemistry parameters consisted of lower platelet counts and higher urea in males at 200 mg/kg/day, higher red cell distribution width in females at 200 mg/kg/day, and higher cholesterol in males and females at 100 and 200 mg/kg/day. Since means of these parameters remained within the internal control range, and had no corroborative morphological lesions, these were considered non-adverse. Higher cholesterol was also recorded for F0-generation males as outlined above.

Higher T4 was recorded at 100 and 200 mg/kg/day in Cohort 1A males and females at PND 89 (1.21x and 1.33x, respectively for males, and 1.41x and 1.58x, respectively for females). Means remained within the internal control range and were not accompanied by test item-related changes in thyroid morphology or TSH levels. Higher T4 and higher TSH was recorded or F0-generation males as outlined above. Since means remained within the internal control data range, these variations were considered not adverse. T4 and TSH levels at PND 4 and PND 21/22 were considered not affected by treatment with the test item.

No treatment-related effects on F1-animals were recorded for other developmental parameters including mortality, weekly arena observations, body weight and food consumption, balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first estrus, time between vaginal opening and first estrus, length and regularity of the estrous cycle, and sperm motility, concentration and morphology, coagulation parameters, urinalysis, macroscopy and organ weights.

Histopathologically, no test-item related effects were noted including on stage-dependent qualitative evaluation of spermatogenesis, ovarian follicle and corpora lutea counts of females of Cohort 1A and on morphology of reproductive organs.

Due to mortality among pups, no Surplus Cohort was added to the study and priority was given to allocate pups to other cohorts. Hence, no spleen and thymus weights were recorded from “up to 10 pups/sex/group” shortly after weaning as per OECD443 testing guideline. Since there were no test-item related effects noted on weight and morphology of these organs in F0 animals and Cohort 1A animals, this was considered not to have adversely affected interpretation of the study results.

TDAR results were considered not affected by treatment with the test item. The incidence of non-responding animals (i.e. anti-KLH IgM levels <LLOQ) was not affected following administration of DGEBADA. Based on the data available from the responding animals, there were no DGEBADA related effects on the levels of anti-KLH IgM antibodies detected at any dose levels Moreover no pathology findings in the lymphoid organs (i.e. histopathology and organ weight) and no test item-related changes in splenic lymphocyte subpopulations, it is confirmed that the test item did not induce any immunotoxic effect.

 

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1, 2 and 3), the following no-observed-adverse-effect levels (NOAEL) and/or No-Observed-Effect-Levels (NOEL) of DGEBADA were established:

> General toxicity (F0): NOAEL of at least 200 mg/kg/day(possible adversity of the higher TSH in males at 40, 100 and 200 mg/kg/dayandhigher total T4 in males at 100 and 200 mg/kg/day could not be assessed within this type of study and was therefore not taken into account when determining the NOAEL for general toxicity). The No Observed Effect Level (NOEL) could not be determined based on the lower body weight gain observed at 200 mg/kg/day in F0 females during lactation, and the high TSH levels in males from 40 mg/kg/day.

> Reproductive toxicity (F0): NOAEL of at least 200 mg/kg/day in absence of adverse effects at this dose on reproduction parameters.

> General toxicity (F1): NOAELof at least 200 mg/kg/day (possible adversity of the higher T4 in Cohort 1A males and females at 100 and 200 mg/kg/day could not be assessed within this type of study and was therefore not taken into account when determining the NOAEL). A No Observed Effect Level (NOEL) of 40 mg/kg/day was determined based on the presence of rales and higher cholesterol at 100 and 200 mg/kg/day in F1 males and females.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is considered to be reliable, it was conducted with OECD guidelines (OECD 443).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Extended one-generation reproduction toxicity study (van Otterdijk 2020)

The objective of this study was to provide an evaluation of the pre- and postnatal effects of DGEBADA on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats. 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 DGEBADA 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. 

Furthermore, the information obtained from the developmental neurotoxicity and developmental immunotoxicity assessments characterized potential effects in those systems.

The dose levels in this study were selected to be 0, 40, 100 and 200 mg/kg/day, based on the results of previously conducted (reproductive) toxicity studies with oral exposure of DGEBADA in rats.

A Lactational Transfer Study was performed prior to this studyto determine if DGEBADA could transfer to mother’s milk after maternal exposure fromDay 6 post-coitum to Day 13 of lactation, inclusive. All measured milk samples collected from dams on Day 13 of lactation revealed values of DGEBA diacrylate (the main compound present in the test item) that were below the lower limit of quantification (LLOQ). This result indicated that DGEBADA is not excreted in the mother’s milk at detectable levels, when administered up to a dose level of 200 mg/kg/day by once daily oral gavage from Day 6 post-coitum until Day 13 of lactation inclusive.

No adverse changes were noted in any of the F0-Generation parameters investigated up to the highest dose level tested (200 mg/kg/day). No mortality and no adverse clinical signs related to treatment occurred during the study period.

Non-adverse changes in body weight were recorded among females, however the lower mean body weight gain of females at 200 mg/kg/day was considered to be treatment-related. Statistically significant lower than controls on Days 14 and 21 of the lactation phase (0.7x and 0.5x respectively). The mean body weight gain at the end of the lactation phase was 0.5x of the control mean. The other variations in mean absolute body weights noted during the premating and post-coitum phases were considered not to be related to treatment.

Food consumption before or after correction for body weight was considered not affected by treatment with the test item in males and females.

Haematological parameters of treated rats were considered not to have been affected by treatment with the test item.

Non-adverse changes in coagulation and clinical chemistry parameters consisted of an elongated prothrombin time and higher cholesterol in males at 200 mg/kg/day are considered to be related to treatment. Since means of these parameters remained within the internal control range, and had no corroborative morphological lesions, these were considered non-adverse.

A trend towards higher thyroid stimulating hormone (TSH) was recorded in males at 40, 100 and 200 mg/kg/day, but means remained within the internal control range. Also, higher total T4 was recorded in males at 100 and 200 mg/kg/day; mean was outside the internal control range at 200 mg/kg/day only. Serum levels of TSH and T4 in F0-females were considered not affected by treatment. These changes in males were not accompanied by test item-related changes in thyroid morphology, but were considered as related to treatment. However, possible adversity of this effect could not be assessed within this type of study. No test item-related changes were noted in any of the other F0-Generation parameters investigated in this study (i.e. functional observations, urinalysis, macroscopic examination, organ weights and microscopic examination).

Reproduction results – F0-generation : No test item-related changes were noted in any of the reproductive parameters investigated up to the highest dose level tested (200 mg/kg/day), i.e. sperm motility, sperm concentration and morphology, mating and fertility indices, precoital time, number of implantations, estrous cycle, 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 test item-related changes were noted in any of the developmental parameters investigated up to the highest dose level tested (200 mg/kg/day) during the gestation and lactation phases. Five animals of the control group, seventeen animals at 40 mg/kg/day, twelve animals at 100 mg/kg/day, and twenty-nine animals at 200 mg/kg/day (including one animal at 40 mg/kg/day and four animals at 200 mg/kg/day that were sacrificed or found dead post-weaning) did not survive the scheduled treatment period. It was considered that this mortality was not related to toxicity of the test item but had occurred principally as a result of the dosing procedure. Clear relationship between the dose and mortality incidence was absent, most cases of mortality occurred on a single day, and the nature of the macroscopic observations was specific for the animals that were found dead/sacrificed and were suggestive of a gavage-related occurrence.

No test item-related changes were noted in any of the following developmental parameters investigated in this study: gestation index and duration, live birth, viability and weaning indices, parturition, sex ratio, litter size, maternal care and early postnatal pup development consisting of mortality, pups’ clinical signs, pups’ body weight, anogenital distance, areola/nipple retention, thyroid hormone levels in pups (T4 of PND 4, T4 and TSH of PND 22) and macroscopic examination of pups sacrificed at the end of the lactation period.

 

Developmental results – F1 generation (post-weaning) : A dose-related but non-adverse incidence of breathing rales was recorded at 100 and 200 mg/kg/day, primarily during the first two weeks of treatment. Incidences were 12/60 males and 12/60 females at 100 mg/kg/day, and 17/60 males and 8/60 females at 200 mg/kg/day. Incidences of rales in males at 40 mg/kg/day (3/60 males) were comparable to incidences in control females (2/60 control females), and as such these may not be attributable to the test item. Since these rales essentially resolved with continuing treatment, this was considered not an adverse finding. However, it could not be excluded that occurrence of these rales may be associated with deaths that occurred primarily prior to weaning, as most of these deaths showed lung lesions (lungs that were not collapsed, and fluid in the thoracic and/or abdominal cavity) suggestive of a gavage-related occurrence.

At 200 mg/kg/day, non-adverse treatment-related changes in acoustic startle response parameters of Cohort 2A males were recorded.These changes consisted of a higher average response amplitude (both as overall mean response (1.38x), and during the first four test blocks (1.64x, 1.38x, 1.42x and 1.42x for blocks 1, 2, 3 and 4, respectively)), maximum response amplitude (both as overall mean (1.23x) and during the first test block (1.47x)) and latency to achieving the maximum response amplitude (both as overall mean (1.27x) and during the first test block (1.46x)). Females at this dose showed no such changes in acoustic startle response parameters, and these parameters were considered not affected by treatment with the test item in males at 40 and 100 mg/kg/day. Other developmental neurotoxicity endpoints of Cohort 2A animals (detailed clinical observations, rectal temperature, hearing ability, pupillary reflex, foot splay, grip strength and motor activity) were considered not affected by treatment, and there was no apparent relationship between body weights and the level of acoustic startle response on an individual animal basis. These changes were considered not adverse since the direction of change consisted of an increased response rather than a decreased response, the auditory reflex loop was functioning at least in a comparable manner as in controls, habituation occurred as expected and recorded for control rats, means were only slightly outside historical control data range for maximum response amplitude in Block 1 and since the changes only occurred for males and not for females.

No test item-related changes were recorded for other developmental neurotoxicity endpoints determined in Cohort 2A and 2B animals, consisting of fixed brain weights, brain dimensions (length and width of brain), routine sections of brain or peripheral nervous system and morphometric analysis of the brain.

Non-adverse changes in haematology and clinical chemistry parameters consisted of lower platelet counts and higher urea in males at 200 mg/kg/day, higher red cell distribution width in females at 200 mg/kg/day, and higher cholesterol in males and females at 100 and 200 mg/kg/day. Since means of these parameters remained within the internal control range, and had no corroborative morphological lesions, these were considered non-adverse. Higher cholesterol was also recorded for F0-generation males as outlined above.

Higher T4 was recorded at 100 and 200 mg/kg/day in Cohort 1A males and females at PND 89 (1.21x and 1.33x, respectively for males, and 1.41x and 1.58x, respectively for females). Means remained within the internal control range and were not accompanied by test item-related changes in thyroid morphology or TSH levels. Higher T4 and higher TSH was recorded or F0-generation males as outlined above. Since means remained within the internal control data range, these variations were considered not adverse. T4 and TSH levels at PND 4 and PND 21/22 were considered not affected by treatment with the test item.

No treatment-related effects on F1-animals were recorded for other developmental parameters including mortality, weekly arena observations, body weight and food consumption, balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first estrus, time between vaginal opening and first estrus, length and regularity of the estrous cycle, and sperm motility, concentration and morphology, coagulation parameters, urinalysis, macroscopy and organ weights.

Histopathologically, no test-item related effects were noted including on stage-dependent qualitative evaluation of spermatogenesis, ovarian follicle and corpora lutea counts of females of Cohort 1A and on morphology of reproductive organs.

Due to mortality among pups, no Surplus Cohort was added to the study and priority was given to allocate pups to other cohorts. Hence, no spleen and thymus weights were recorded from “up to 10 pups/sex/group” shortly after weaning as per OECD443 testing guideline. Since there were no test-item related effects noted on weight and morphology of these organs in F0 animals and Cohort 1A animals, this was considered not to have adversely affected interpretation of the study results.

TDAR results were considered not affected by treatment with the test item. The incidence of non-responding animals (i.e. anti-KLH IgM levels <LLOQ) was not affected following administration of DGEBADA. Based on the data available from the responding animals, there were no DGEBADA related effects on the levels of anti-KLH IgM antibodies detected at any dose levels Moreover no pathology findings in the lymphoid organs (i.e. histopathology and organ weight) and no test item-related changes in splenic lymphocyte subpopulations, it is confirmed that the test item did not induce any immunotoxic effect.

 

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1, 2 and 3), the following no-observed-adverse-effect levels (NOAEL) and/or No-Observed-Effect-Levels (NOEL) of DGEBADA were established:

> General toxicity (F0): NOAEL of at least 200 mg/kg/day(possible adversity of the higher TSH in males at 40, 100 and 200 mg/kg/dayandhigher total T4 in males at 100 and 200 mg/kg/day could not be assessed within this type of study and was therefore not taken into account when determining the NOAEL for general toxicity). The No Observed Effect Level (NOEL) could not be determined based on the lower body weight gain observed at 200 mg/kg/day in F0 females during lactation, and the high TSH levels in males from 40 mg/kg/day.

> Reproductive toxicity (F0): NOAEL of at least 200 mg/kg/day in absence of adverse effects at this dose on reproduction parameters.

> General toxicity (F1): NOAEL of at least 200 mg/kg/day (possible adversity of the higher T4 in Cohort 1A males and females at 100 and 200 mg/kg/day could not be assessed within this type of study and was therefore not taken into account when determining the NOAEL). A No Observed Effect Level (NOEL) of 40 mg/kg/day was determined based on the presence of rales and higher cholesterol at 100 and 200 mg/kg/day in F1 males and females.

Combined repeated dose and reproduction/ developmental screening test (Stannard 2010)

In a GLP study conducted according to OECD guideline 422, three groups each comprising five male and five female rats for the Toxicity subgroup and five male and ten female rats for the Reproductive subgroup received 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid at doses of 100, 300 or 900 mg/kg bw/day. Toxicity subgroup males and females and Reproductive subgroup males were treated daily for five consecutive weeks. Reproductive subgroup females were treated daily for two weeks before pairing, throughout pairing, gestation and lactation until the day prior to termination on Day 7 of lactation. A similarly constituted Control group received the vehicle, propylene glycol, at the same volume-dose.

During the study, data was recorded on clinical condition, performance under detailed physical and arena examination, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, water consumption (visual), haematology, blood chemistry, oestrous cycles, mating performance and fertility and gestation length. Organ weight, macroscopic and microscopic pathology investigations were undertaken in the adults. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken.

There was only one mortality during the course of the study, therefore considered unrelated to treatment. There were no signs observed among treated males and females at routine physical examination or during the arena observations that were considered to be related to treatment. There was no effect of treatment on the food consumption, sensory reactivity findings, grip strength values and motor activity in males and females throughout the study.

At 900 mg/kg bw/day, mean bodyweight gain of males was slightly lower than Control during the first week of treatment. During the second week, weight gain for these males remained slightly (but not significantly) lower than Control, but thereafter, mean weight gain was essentially similar to Control in all treated groups, and the bodyweight gain of females during gestation and lactation was unaffected by treatment at all dose levels investigated.

Haematological assessment revealed slightly prolonged prothrombin time among males and females receiving 900 or 300 mg/kg bw/day. Biochemical changes in the blood plasma comprised high total bilirubin and total bile acid concentrations in males receiving 900 or 300 mg/kg bw/day and females receiving 900 mg/kg bw/day, and high cholesterol concentrations in males and females in all treated groups. A dose-related trend was evident for all of these haematological and biochemical parameters.

There was no clear evidence of an adverse effect of treatment on mean organ weights among all animals at scheduled termination. There were no macroscopic abnormalities and no test substance-related lesions at microscopic examination.

Oestrous cycle length, pre-coital interval, mating performance and fertility and gestation length of the Reproductive subgroup females were unaffected by treatment. There were no clinical signs observed for F1 offspring that were considered to be related to parental treatment and offspring survival and growth from birth to Day 7 of age was unaffected by treatment. There were no macroscopic abnormalities detected among the offspring that died during the early post-natal period, or at scheduled termination on Day 7 of age that were attributable to parental treatment.

Based on the results of this study, it was concluded that the NOAEL for systemic toxicity in the CD rat following 5 weeks of treatment was higher than 900 mg/kg bw/day. The NOAEL for the reproductive/developmental toxicity screening test within the scope of this study was also concluded to be higher than 900 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

In the developmental toxicity studies performed in rats and rabbits, no adverse effects were observed in dams and fetus.

In conclusion based on these results, no the NOAEL for maternal toxicity and the NOAEL for prenatal developmental toxicity were defined as 1000 mg/kg bw/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 21-Oct-2014 to 24-Apr-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22nd January 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

EC 2004/73 B31, dated April 29, 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

EPA 712-C-98-207, dated August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines (Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology (2-1-18), Agricultural Production Bureau, dated November 24, 2000).

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: At least 11 weeks at day 0 post coitum
- Weight at study initiation: 173 to 261 g at day 0 post coitum
- Fasting period before study: None
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK and ISO-BLOX from Harlan Laboratories B.V. / Netherlands).
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 13/14) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed was analyzed for contaminants.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. Bacteriological assay, chemical and contaminant analyses of respective samples were performed.
- Acclimation period: 7 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.

IN-LIFE DATES: From: 21-Oct-2014 (start of 7-day Acclimatization) To: 27-Nov-2014 (Last Necropsy)

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

300

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared at least once weekly using the test item as supplied by the Sponsor.
Vehicle was pre-warmed to a temperature of approximately 40 °C. DGEBADA was weighed into a glass beaker on a tared precision balance and approximately 80 % of the warm vehicle was added (w/v). The formulations were mixed with a magnetic stirrer until a homogeneous mixture was obtained. Having obtained a homogeneous mixture, the remaining vehicle was added until the required final volume and thereafter stirred for approximately 15 minutes. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Stability and Storage of Dose Formulations: For at least 10 days at room temperature (20 ± 5 °C) based on stability results of Harlan Laboratories Study no. D94595 (Braun 2015, subchronic repeated oral toxicity

DIET PREPARATION
- Not relevant due to exposure via gavage

VEHICLE
- Justification for use and choice of vehicle: Considered non-toxic to the test animals and ability to form a homogeneous mixture with the test item
- Concentration in vehicle (nominal): 0, 25, 75 and 250 mg/mL (at 0, 100, 300 and 1000 mg/kg bw/day, respectively)
- Amount of vehicle (by gavage): 4 mL/kg body weight with a daily adjustment to the actual body weight
- Lot/batch no.: BCBK9989V (Source: Sigma-Aldrich Chemie GmbH, Steinheim / Germany, Expiry Date: 20-May-2016)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Representative samples were dispatched to the analytical laboratories internally (at room temperature) and stored frozen at -20 ± 5 °C until analysis.
The test item was used as the analytical standard.
Stock solutions of DGEBADA in acetonitrile were prepared for external calibration. For example, 31.12 mg of DGEBADA was weighed into a 50 mL volumetric flask and filled to about 75 % of final volume with acetonitrile. The mixture was sonicated for at least five minutes and brought to volume with acetonitrile to yield a solution with a concentration of 622.4 µg/mL. Aliquots of this stock calibration solution were diluted with acetonitrile to obtain calibration solutions with nominal concentrations ranging from 10.58 to 200.4 µg/mL. On each occasion calibration solutions derived from two stock solutions were used for calibration.
Each sample was transferred into an appropriate volumetric flask. The sample vial was successively rinsed with at least two portions of acetonitrile and the rinsings were combined in the volumetric flask. The flask was filled to about 75 % of the target volume with acetonitrile and dissolution was achieved by sonication for at least five minutes. The flask was filled to the mark with acetonitrile. Where necessary, sample solutions were further diluted with acetonitrile into the calibration range.
Typical HPLC systems from Merck-Hitachi series 7000 were used:
- Computerized System: EZ Chrom Elite; Agilent Technologies
- Column: Kinetex C18; 50 x 4.6 mm, 5 µm
- Pre-Column: Phenomenex C18; 4 x 3 mm
- Column Temperature: Ambient
- Eluent A: Purified water / acetonitrile (50/50, v/v)
- Eluent B: Acetonitrile
- Gradient:
Time [min]; Eluent A [%]; Eluent B [%]
0; 100; 0
4; 100; 0
8; 5; 95
10; 5; 95
10.1; 100; 0
15; 100; 0
- Flow: 1 mL/min
- Wave Length: 230 nm
- Injection Volume: 10 µL
The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and calibration solution concentrations. All calibration points used met the acceptance limit of ±20 % variation from the calibration curve derived by linear regression analysis. The coefficients of determination (R²) were higher than 0.99.
The DGEBADA concentrations in the analysed dose formulations ranged from 86.0 to 102.0 % (dates of Preparation of the stock solution 03-Nov-2014, Day 6 and 14-Nov-2014, Day 17) with reference to the nominal and were within the accepted range of ±20 %. The homogeneous distribution of DGEBADA in the preparations was approved because single results found did not deviate more than 7.3 % from the corresponding mean and met the specified acceptance criterion of =15 %. Accordingly the effects were assigned to the nominal concentrations.

Details on mating procedure:

- Impregnation procedure: Cohoused, After acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed. The day of mating was designated day 0 post coitum.
- M/F ratio per cage: 1:1
- Length of cohabitation: After 10 days pairing of all test animals was successfully completed.
- Further matings: No
- Verification of same strain and source of both sexes: Yes, all animals belonged to the RccHan™: WIST(SPF)
- Proof of pregnancy: Vaginal plug observed or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: None

Duration of treatment / exposure:

15 days (during gestation period from day 6 to 20 post coitum)

Frequency of treatment:

Daily, at approximately 24 hour intervals

Duration of test:

21 days post coitum
Experimental Starting Date (Delivery of Animals): 21-Oct-2014
Acclimatization: 6 days from 21-Oct-2014 (Day -7)
Initiation of Pairing: 27-Oct-2014 (Day -1)
First Test Item Administration: 03-Nov-2014 (Day 6)
First Necropsy (Caesarean Section): 18-Nov-2014 (Day 21)
Termination (Last Necropsy): 27-Nov-2014 (Day 30)
Experimental Completion Date: 24-Apr-2015

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

24 mated females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous non-GLP dose range-finding study in Han Wistar rats, Harlan Laboratories non-GLP study no. D83997, using dose levels of 0, 100, 300 and 1000 mg/kg/day.
- Rationale for animal assignment: Computer-generated random algorithm

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Viability / Mortality observation were made twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Daily from day 0 until day 21 post coitum.

FOOD CONSUMPTION: Yes
- Time schedule: For the following periods: days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 21 post coitum

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 21 post coitum
- Organs examined: Gross macroscopic examination of all internal organs

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Post mortem examination with emphasis on the uterus, uterine contents, corpora lutea count and position of foetuses in the uterus was performed and the data recorded.
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions (embryonic): Yes
- Number of late resorptions (foetal): Yes
- Other: The uteri (and contents) of all females with live foetuses were weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.

Fetal examinations:

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

Foetuses were removed from the uterus by caesarean section, sexed, weighed individually, examined for gross external abnormalities, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
1. Microdissection technique (sectioning/dissection technique): Approximately one half of the foetuses from each litter were fixed in Bouin's fixative. They were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissue was preserved in a solution of glycerin/ethanol (one foetus per container). Descriptions of any abnormalities and variations were recorded.
2. The remaining foetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. After fixation in ethanol, carcasses were processed through solutions of glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage. The skeletons were examined and all abnormal findings and variations were recorded. The assessment included, but was not limited to all principal skeletal structures including cranium, vertebral column, rib cage and sternum, pectoral and pelvic girdles. The specimens were preserved individually in small containers.

The foetuses were sent to the contributing scientist for peer review at Huntingdon Life Sciences.

Statistics:

The following statistical methods were used to analyse food consumption, body weights, reproduction and skeletal examination:
• Means and standard deviations of various data were calculated and included in the report.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied, if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied, if the variables could be dichotomized without loss of information.

Indices:

No indices were calculated as percentages were used.

Historical control data:

Tables summarizing the available historical control data from six studies performed at the test site (referenced 12/02, 12/03, 12/04, 13/01, 13/02 and 13/03) are given in Appendix V of the study report.
For the interpretation of results regarding Ribs (Supernumerary cervical, Rudimentary) the historical data record was used. It showed occurrences in studies 12/02, 12/04, 13/01 and 13/02, while such effects lacked in study 12/03 and were not examined in study 13/03 (data shown on page 329 of the study report):
In foetuses the numbers and the related percentages were: 1 (1 %), 1 (1 %), 1 (1 %) and 3 (2 %)
In litters the numbers and the related percentages were: 1 (5 %), 1 (4 %), 1 (5 %) and 2 (10 %)
Furthermore foetal body weight gain and non-ossification data were used for data interpretation of this study, but they were due to their complexity not reproduced here.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical symptoms or signs were observed at any dose during the study. Female no. 54 (group 3) had vaginal bleeding, showed ruffled fur and decreased activity, and was in a general weakened condition on day 20 post coitum. These symptoms were not considered to be test item-related, because they were restricted to this single animal in this group and no clinical signs were observed in animals of group 4.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Female no. 54 (group 3) was sacrificed on day 20 post coitum. The animal had vaginal bleeding, showed ruffled fur and decreased activity, and was in a general weakened condition. All other females survived until the scheduled necropsy.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No effects on mean absolute body weights were recorded. T
ransiently statistically significant changes in body weight gain were observed in the group 2 on Day 14, and in the group 4 on Day 9 and from Day 12 to 16; there were of minimal magnitude and considered to be incidental findings.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean daily food consumption was similar in all groups from days 0-6 post coitum. There were no test item-related changes in food consumption during days 6-21 post coitum.

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 specified

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):

No test item-related findings were observed at any dose level during macroscopic examination. Pale discoloured lungs which were reduced in size, spleen reduced in size, a clay-coloured liver, and a haemorrhagic-watery fluid containing stomach were recorded in female no. 54 (group 3) which was sacrificed on day 20 post coitum in moribund condition.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

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:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Placenta Weights:
In group 4, placenta weights on a litter basis were increased by 23 and by 24 % on an individual basis. This increase was considered to be test item-related. Slight, but statistically increased placenta weights on an individual basis in groups 2 and 3 were not considered to be test item-related, because the increase was only minor (+8 and +6.5 %, respectively) and not dose-related.
Mean placenta weights calculated on a litter basis were: 0.484 g, 0.515 g, 0.510 g and 0.594 g whereas calculated on an individual basis, they were 0.476 g, 0.514 g, 0.507 g and 0.589 g, both cited in order of ascending dose level.

Details on maternal toxic effects:

The statistically significant decrease in the number of implantation sites and a decrease of the pre-implantation loss in all test item-treated groups were not test item-related since this occurs before treatment starts.
The relevant reproduction data (mean post-implantation loss and mean number of foetuses per dam) were not affected by treatment with the test item.

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

In group 4, placenta weights on a litter basis were increased by 23 and 24 % on litter and individual bases, respectively, and considered to be test item-related. Differences noted in groups 2 and 3 were unrelated to dose and therefore considered to be incidental.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): No test item-related effects on mean body weights of live foetuses were recorded.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratios were similar to those of the controls.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

No test findings were observed during external examination of the foetuses.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No test item-related findings were noted during external and fresh visceral examinations of foetuses or during the skeletal examination of the foetuses.
The incidence of incomplete or non-ossification was slightly elevated in a small number of bones: Most of these values remained within the ranges of the respective historical control values. A small number of supernumerary ribs remained within the ranges of the respective historical control values and are therefore considered to be of no toxicological relevance.

Visceral malformations:

no effects observed

Description (incidence and severity):

No test item-related findings were noted during external and fresh visceral examinations of foetuses or during the skeletal examination of the foetuses.

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

No test item-related findings were noted during external and fresh visceral examinations of foetuses or during the skeletal examination of the foetuses.In some cases, these values exceeded the historical control data ranges but were considered to be specific structures that typically vary in development. These findings are unlikely to be related to the treatment and therefore considered to be of no toxicological relevance.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

In conclusion based on these results, the NOEL (No Observed Effect Level) and NOAEL (No Observed Adverse Effect Level) for maternal toxicity was considered to be above 1000 mg/kg bw/day. Placenta weight differences seen in group 4 were considered to be test item-related changes; the NOEL for prenatal development was therefore considered to be 300 mg/kg/day. The NOAEL for prenatal developmental toxicity was defined as 1000 mg/kg bw/day.

Executive summary:

The oral (gavage) prenatal developmental toxicity of the test item 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid (CAS 55818-57-0, DGEBADA) to Wistar rats was investigated in a GLP-compliant dose-effect study according to the OECD TG 414 (2001).

The purpose of this study was to detect effects on the pregnant rat and development of the embryo and foetus consequent to exposure of the female to the test item from day 6 post coitum (implantation) to day 20 post coitum (the day prior to caesarean section). Four groups of 24 mated females per group were treated by gavage with DGEBADA once daily at nominal dose levels of 0 (control group), 100, 300 and 1000 mg/kg body weight/day (named groups 1 to 4, respectively). A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (PEG 300). All females were sacrificed on day 21 post coitum and the foetuses were removed by caesarean section.

Regarding the maternal data, no test item-related premature deaths were recorded. One female in group 3 was sacrificed for ethical reasons unrelated to the treatment with the test item. No test item-related clinical symptoms or signs, no changes in the food consumption and no effects on mean absolute body weights and body weight gain were observed at any dose during the study. The relevant reproduction data (post-implantation loss and number of foetuses per dam) were not affected by treatment with the test item and no test item-related macroscopic findings were observed at any dose level during macroscopic examination.

The foetal examinations revealed in group 4 placenta weights on a litter basis being increased by 23 and by 24 % on an individual basis. This increase was considered to be test item-related. Differences noted in groups 2 and 3 were unrelated to dose and therefore considered to be incidental. No test findings were observed during external examination of the foetuses. No test item-related effects on sex ratio and mean body weights of the foetuses were noted in any group. No test item-related abnormalities and variations were noted during external and fresh visceral examinations of foetuses and no findings were noted during skeletal examination of the foetuses. A small number of incomplete or non-ossified bones and/or supernumerary ribs remained within the ranges of the respective historical control values and were therefore considered to be of no toxicological relevance. There were no additional cartilage finding in any foetus that was considered to be related to the treatment with the test item.

In conclusion based on these results, the NOEL (No Observed Effect Level) and NOAEL (No Observed Adverse Effect Level) for maternal toxicity was considered to be above 1000 mg/kg bw/day. Placenta weight differences seen in group 4 were considered to be test item-related changes; the NOEL for prenatal development was therefore considered to be 300 mg/kg/day. The NOAEL for prenatal developmental toxicity was defined as 1000 mg/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 May 2017 - 06 July 2017

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:

22 January 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Centre LAGO (Vonnas, France)
- Age at the beginning of the treatment period: approximately 18-20 weeks old on the day of treatment
- Mean body weight at the beginning of the treatment period: 3681 g (range: 3215 g to 4170 g)
- Fasting period before study: no
- Housing: The animals were individually housed in noryl cages (Tecniplast, 4200 cm2).
- Diet: (SAFE, Augy, France) pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of 4 or 5 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 5 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 8h/16h

IN-LIFE DATES: 29 May 2017 to 06 July 2017

Route of administration:

oral: gavage

Vehicle:

other: methylcellulose (1%) in drinking water treated by reverse osmosis

Details on exposure:

PREPARATION OF DOSING FORMULATIONS:
- Emulsion in the vehicle
- Concentration in vehicle: 33.3, 100 and 333 mg/mL
- Amount of vehicle (if gavage): 3 mL/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: High Performance Liquid Chromatography with UV detection (HPLC-UV)
Test item concentrations: within an acceptable range of variation (-10.4% to -2.7%) when compared to the nominal values (± 15% required).
Homogeneity: Dose formulations ranging from 2 mg/mL to 333.3 mg/mL are therefore considered to be suitable for routine administration in GLP Toxicological studies within the day of the preparation.
Stability: not assessed, dose formulation prepared daily.

Details on mating procedure:

The females were mated at the breeder's facilities. The day of confirmed mating (visual assessment) was designated as Day 0 p.c.

Duration of treatment / exposure:

The dose formulations were administered daily from Days 6 to 28 p.c., inclusive.

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

24 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected in agreement with the Sponsor, on the basis of the results of a preliminary study where time-mated New Zealand White female rabbits were given the test item by gavage from Days 6 to 28 p.c. at 100, 300 or 1000 mg/kg/day.
. In this study, there were no apparent test item-related effects at 100 and 300 mg/kg/day. At 1000 mg/kg/day, there were no mortality, no test item-related clinical signs and no obvious effects at necropsy. One pregnant female had a food consumption = 10 g/day from Day 15 p.c. and lost 18% of body weight between Days 12 and 29 p.c. Two other pregnant females had a food consumption lower than 50 g/day at the end of the study and lost concomitantly 4 or 7% of body weight. Mean fetal body weight was lower in their litter. There were no such effects on food consumption, body weight and fetal body weight in the fourth pregnant female.
In absence of death and severe suffering and in order to induce some maternal toxicity at the high-dose at least, as requested by the guideline, the same dose levels were selected for the present study.

- Rationale for animal assignment: stratified procedure.

Maternal examinations:

MORTALITY/MORBIDITY:
- Time schedule: each animal was checked for mortality or signs of morbidity once a day before the treatment period and at least twice a day during treatment period, including weekends and public holidays.
One female from group 4, showing signs of poor clinical condition, was weighed and humanely euthanized.

CLINICAL SIGNS:
- Time schedule: from arrival, the animals were observed once a day as part of routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time of day, for the recording of clinical signs.

BODY WEIGHT:
- Time schedule: the body weight of each female was recorded on Days 2, 4, 5, 6, 9, 12, 15, 19, 24 and 29 p.c. and prior to premature euthanasia.

FOOD CONSUMPTION:
- Time schedule: The quantity of food consumed by each animal was recorded for the following intervals: Days 2-4, 4-5, 5-6, 6-9, 9-12, 12-15, 15-19, 19-24 and 24-29 p.c.

POST-MORTEM MACROSCOPIC EXAMINATION:
- Sacrifice on Day 29 post-coitum.
- Examined: principal thoracic and abdominal organs.

Ovaries and uterine content:

The ovaries and uterine content were examined after termination, including:
- Gravid uterus weight,
- Number of corpora lutea,
- Number and distribution of dead and live fetuses,
- Number of implantation sites,
- Number of early resorptions,
- Number of late resorptions,
- Number of uterine scars and evaluation of placentas.
The placenta weight was recorded for each live fetus and the fetal weight/placental weight ratio was calculated.

Fetal examinations:

- External examinations: Yes: all fetuses per litter
- Soft tissue examinations: Yes: all fetuses per litter
- Skeletal examinations: Yes: all fetuses per litter
- Head examinations: Yes: half fetuses per litter
- Other: body weight, sex.

Statistics:

Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).

Indices:

% Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites

Historical control data:

Cf attached document

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Se table 1.
The clinical signs recorded during the treatment period in pregnant animals were recorded in isolated animals and/or without dose-relationship. They were therefore considered to be not test item-related.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

One female treated at 300 mg/kg/day was found dead on Day 12 p.c. This female was difficult to administer during the 3 days before, and blood was found on the gavage tube on the eve. At necropsy, reddish color of the left lung was observed and the female had only late resorptions in the uterus. This death was considered to be related to the gavage procedure.
Another female treated at 1000 mg/kg/day was prematurely euthanized on Day 23 p.c. following abortion (placentas and fetus in the bedding); emaciated appearance and absence of feces were recorded for this female for 6 or 8 days before abortion. From treatment initiation, its food consumption was near or at 0 g/day and concomitant to body weight losses (-16% between Days 6 and 19 p.c.). At necropsy, thickened pyloric mucosa, reddish content in the vagina and dilatation of the gall bladder were observed.
A relationship between this abortion and the test item treatment could not be excluded as it happened at the high-dose.
There were no other unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See table 2.
At 1000 mg/kg/day, there were no test item treatment-related effects on mean body weights. However, on Days 6 to 12 p.c. females had a mean body weight loss followed by a low mean body weight gain. Mean body weight change was then comparable with controls from Day 12 to 24, but at the end of the treatment period there were more females losing weight (<10%; generally associated with reduced food consumption) than in controls.
These effects on mean body weight change were considered to be test item-related and non-adverse (slight and/or reversible effect, no toxicological impact on mean body weights).
At 100 and 300 mg/kg/day, there were no test item treatment-related effects.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See table 3.
At 1000 mg/kg/day and when compared with controls, mean food consumption was statistically significantly lower on Days 6 to 15 p.c. At the end of the treatment, food consumption was strongly reduced (associated with body weight loss) in more females when compared with controls.
These effects were considered to be test item-related and non-adverse (no severe differences from controls; no toxicological impact on mean body weights).
At 100 and 300 mg/kg/day, there were no test item treatment-related effects.

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:

effects observed, non-treatment-related

Description (incidence and severity):

None of the findings recorded in surviving pregnant females were considered to be test item-related (no dose-relationship).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

See table 4.
Gravid uterus weight and net body weight change
There were no test item-related effects on mean carcass weight and mean net body weight change.

At 100 and 1000 mg/kg/day when compared with controls, mean uterus weight was statistically significantly lower. This finding was considered to be due mainly to the lower number of fetuses in both groups (not test item-related).

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

See table 5.
One female treated at 1000 mg/kg/day was prematurely euthanized on Day 23 p.c. following abortion (placentas and fetus in the bedding); emaciated appearance and absence of feces were recorded for this female for 6 or 8 days before abortion. From treatment initiation, its food consumption was near or at 0 g/day and concomitant to body weight losses (-16% between Days 6 and 19 p.c.). At necropsy, thickened pyloric mucosa, reddish content in the vagina and dilatation of the gall bladder were observed.
A relationship between this abortion and the test item treatment could not be excluded as it happened at the high-dose.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

See table 5.
There were no effects on mean hysterectomy data considered to be test item-related.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

See table 5.
There were no effects on mean hysterectomy data considered to be test item-related.
At 100 mg/kg/day when compared with controls and historical control data, mean number of live fetuses per litter was lower, reaching statistical level. This was due to high mean rate of pre-implantation loss (therefore not test item-related as treatment started at implantation), as well as to high mean post-implantation loss percentage and mean number of resorptions (considered to be incidental in absence of dose-relationship and as within historical control data).
At 1000 mg/kg/day when compared with controls and historical control data, mean number of live fetuses per litter was lower also. It was considered to be due to the high mean rate of pre-implantation loss (therefore also not test item-related).

Dead fetuses:

effects observed, non-treatment-related

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

See table 5.
On Day 29 p.c., there were 23/24, 22/24, 19/23 and 23/23 pregnant females with live fetuses in control, 100, 300 and 1000 mg/kg/day groups, respectively.
At 300 and 1000 mg/kg/day, the two prematurely euthanized/dead females were pregnant.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

See table 6.
There were no toxicologically significant differences in mean data between groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no test item-related effects on mean percentage of male fetuses per litter (mean data comparable between groups and close to 50%).

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related external variations and malformations in litters.
The only fetal external variations (paw or limb hyperflexion) were mostly recorded in the control group, and the only fetal external malformations (bent tail or short + narrowed tail) were recorded in one fetus of the control group (associated with fused caudal vertebrae) or in one fetus of the 100 mg/kg/day group (associated with fused and absent caudal vertebrae) only.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

See tables 8 to 10.
In test item groups, all cartilages of bones were present or with a correct shape, with a few exceptions: fused, bipartite and misshapen cartilages of sternebrae. For both latter findings, the incidences were isolated and/or similar with controls (no test item-related effects therefore). For the fused cartilages of sternebrae, see discussion on the malformation fused sternebrae below.
At 1000 mg/kg/day and when compared with controls, there were a few skeletal variations (incomplete ossification of interparietal, 1st to 4th sternebrae and 1st metacarpal; thickened ribs) with higher incidences. However, the differences from controls were slight, and/or the incidences were either within or close to the upper limit of the Historical Control Data.
At 300 mg/kg/day, there were 3 fetuses from 3 different litters with unossified pubis, while no control fetus and rare historical control data fetuses had the same findings. Nevertheless this finding was not observed in the other test item groups and the incidence was slight. The incidences of incompletely ossified interparietal were lower/close to the historical control data maximum.
A test item treatment effect was therefore uncertain (especially for thickened ribs at the high-dose whose incidence was slightly higher than Historical Control Data) for the above mentioned variations, but considered of to be non-adverse as they are variations (not malformations), were noted in absence of toxicologically significant effects on mean fetal weight and as litter incidence were generally within Historical Control Data.
The other fetal skeletal variations in test item groups (including extra sternebral ossification site, ossification point on 13th thoracic vertebra and dumbbell ossification of centrum at 1000 mg/kg/day) were noted with incidences isolated or comparable/lower to controls and/or historical control data, and/or with no dose-relationship and statistical significance. They were considered to be incidental or of no toxicological significance.
At 300 and 1000 mg/kg/day, fetal and litter incidences of interparietal or parietal split were higher than in controls or Historical Control Data; a test item treatment effect cannot be excluded. However, these findings were not considered to be adverse in view of the slight incidences and slight differences from controls (no statistical differences), since they do not impact the global shape of the skull and since they are probably due to ossification delay.
Fused sternebrae was present in test item-treated groups at higher fetal and litter incidences than in controls. However, the incidences were generally within historical control data and there were no dose-relationship. A test item treatment effect was considered to be unlikely.
The other fetal skeletal malformations in test item groups were noted with incidences isolated or comparable/lower to controls and/or historical control data and/or with no dose-relationship or statistical significance. They were considered to be incidental.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

See table 7.
At 1000 mg/kg/day when compared with controls, there were higher litter and fetal incidences of findings on the gall bladder. Indeed, 8 fetuses among 6 litters (vs. 2 fetuses among 2 litters in controls) had a morphology finding on their gall bladder (small, bilobed, enlarged, with colored nodule). These findings were considered to be test item-related and non-adverse in view of the slight incidence and as these findings are variations.
At 300 and 100 mg/kg/day, there were higher litter incidences of absent innominate artery than control data and historical control data maximum. In absence of clear dose-relationship, and taking into account that fetal incidences were within historical control data and that the litter incidence at 1000 mg/kg/day was close to the historical control data maximum, an effect of the test item treatment was considered to be unlikely.
The statistical significantly higher fetal incidence of short innominate artery was not attributed to the test item treatment (close to historical control data maximum, no dose-relationship).
The other fetal visceral variations in test item groups, including the ovary serous cyst and the liver colored nodule at 1000 mg/kg/day, were noted with incidences isolated and/or comparable to controls and/or historical control data and/or with no dose-relationship or statistical significance. They were considered to be incidental.
There were no fetal visceral malformations ascribed to the test item treatment.
The only fetal visceral malformations (marked dilated cerebral ventricle, absent aortic arch) were observed in 1 or 2 fetuses of isolated litters from the 100 and 300 mg/kg/day.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 1: Clinical signs

 

Dose level (mg/kg/day)	0	100	300	1000
Emaciated appearance		1
Absence of feces				1
White coloured feces				1
Soiled urogenital area		1
Cutaneous lesions on head		1
Reddish vaginal discharge	1
Blood in the bedding	1	1	1	1
Abnormal growth of teeth			1
Number of affected animals	1/24	2/24	2/23	4/23

Table 2: Body weight

 

Dose level (mg/kg/day)	0	100	300	1000
Mean body weight
Day 6p.c	3693	3659	3657	3694
		-1	-1	0
Day 9p.c	3725	3701	3687	3677
		-1	-1	-1
Day 12p.c	3795	3754	3753	3700
		-1	-1	-3
Day 15p.c	3889	3839	3828	3783
		-1	-2	-3
Day 19p.c	3951	3881	3886	3856
		-2	-2	-2
Day 24p.c	4049	3937	4016	3989
		-3	-1	-1
Day 29p.c	4080	3991	4060	3970
		-2	0	-3
Mean body weight change
Days 6 - 9p.c.	+32	+41	+30	-16*
Days 9 - 12p.c.	+71	+54	+63	+23*
Days 12 - 15p.c.	+93	+85	+75	+83
Days 15 - 19p.c.	+62	+42	+57	+74
Days 19 - 24p.c.	+98	+55	+130	+105
Days 24 - 29p.c.	+31	+54	+44	-18
Days 6 - 29p.c.	+387	+331	+399	+283

In italic, percentage difference (%)vs. controls;Statistical significancevs. controls:*: p<0.05.

Table 3: Food consumption

 

Dose level (mg/kg/day)	0	100	300	1000
Days 6 - 9p.c.	164	168 +2	156 -7	119# -27
Days 9 - 12p.c.	175	185 +6	172 -2	131# -25
Days 12 - 15p.c.	165	171 +4	160 -3	131* -21
Days 15 - 19p.c.	188	181 -4	177 -6	163 -13
Days 19 - 24p.c.	160	168 +5	163 +2	160 0
Days 24 - 29p.c.	107	118	113	90
		+10	+6	-16

In italic, percentage difference (%)vs. controls; Statistical significancevs. controls:*: p < 0.05, #: p < 0.001.

Table 4: Gravid uterus

 

Dose level (mg/kg/day)	0	100	300	1000
Gravid uterus weight	526	430* -18	487 -7	425** -19
Carcass weight	3555	3561	3573	3545
Net weight change from Day 6p.c.	-139	-98.3	-87.5	-142

Statistical significance:*: p < 0.05, **: p < 0.01; data rounded to 3 significant figures when applicable;in italic, percentage difference (%)vs.controls.

Table 5: Hysterectomy data

Dose level (mg/kg/day)	0	100	300	1000	HCD
Number of females with live fetuses	23	22	19	23	159
Mean number ofcorpora lutea	11.8	11.3	11.5	11.3	[11.4-12.8]
Mean number of implantation sites	10.8	9.3	10.3	9.1	[9.5-11.4]
Mean pre-implantation loss (%)	8.8	18.1	10.5	20.1	[10.5-17.9]
Mean number of live fetuses	10.0	7.5**	9.4	8.2	[8.5-10.6]
Mean number of early resorptions	0.3	1.0	0.3	0.7	Late + early [0.36-1.50]
Mean number of late resorptions	0.5	0.8	0.5	0.3
Mean post-implantation loss (%)	6.2	20.1*	7.9	9.2	[3.9-20.5]

Statistical significance:*: p < 0.05 **: p < 0.01.

HCD: historical control data (2015-2016; New Zealand White rabbits): study means [minimum-maximum].

 

Table 6: Fetal body weight and placental weight

 

Dose level (mg/kg/day)	0	100	300	1000	HCD
Mean fetal body weight	35.7	38.2 +7	34.7 -3	34.4 -4	[34.6-39.3]
Mean placental weight	5.26	5.80 +10	5.27 0	5.40 +3	-
Mean ratios of fetal weight/placental weight^a	6.89	6.68	6.75	6.47	-

In italic, percentage difference (%)vs. controls.

a: calculated with Excel, no statistics performed.

HCD: Historical Control Data (2015-2016; New Zealand White rabbits): study means [minimum-maximum].
-: not presented in HCD.

Table 7: Soft tissue variations

Dose-level (mg/kg/day)	0	100	300	1000	HCD
Number of litters	23	22	19	23	159
Number of fetuses	231	164	179	188	1558
Gall bladder
Small gall bladder, F (L)	0.4 (4.3)	1.8 (9.1)		1.1 (8.7)	0.6 (5.6)
Bilobed gall bladder, F (L)				1.1 (8.7)	0.6 (5.6)
Colored nodule on gall bladder, F (L)	0.4 (4.3)			1.1 (8.7)	0.4 (4.0)
Enlarged gall bladder, F (L)				1.1 (4.3)	0.5 (5.3)^a
Liver
Colored nodule				0.5 (4.3)	0.4 (4.0)
Ovary
Serous cyst				0.5 (4.3)	0.6 (5.6)
Vessels
Short innominate artery, F (L)	19.0 (87.0)	28.7* (86.4)	21.8 (73.7)	26.1 (82.6)	26.8 (94.4)
Absent innominate artery, F (L)	9.5 (56.5)	14.0 (63.6)	14.5 (84.2)	16.0 (73.9)	17.9 (72.0)
Litters with visceral variations, n (%)	21 (91.3)	21 (95.5)	18 (94.7)	22 (95.7)	143 (89.9)
Fetus with visceral variations, n (%)	71 (30.7)	75** (45.7)	74* (41.3)	86** (45.7)	481 (30.9)

F: fetal incidence; L: litter incidence,n: number; Statistical significance:*: p < 0.05, **: p < 0.01.

HCD: Historical Control Data (2015-2016; New Zealand White rabbits): maximal study fetal incidence (maximal study litter incidence) for the findings; -: none in HCD.

Table 8: Cartilage examinations

 

Dose-level (mg/kg/day)	0	100	300	1000		HCD
Number of litters	23	22	19	23		159
Number of fetuses	231	164	179	188		1558
Sternebrae
Fused cartilages	1.3 (4.3)	1.2 (9.1)	2.2 (10.5)		1.1 (8.7)	1.8 (16.7)
Bipartite cartilage			0.6 (5.3)			-
Misshapen	0.4 (4.3)	0.6 (4.5)			0.5 (4.3)	-
Litters with cartilage findings, n (%)	22 (95.7)	20 (90.9)	19 (100)		23 (100)
Fetuses with cartilage findings, n (%)	104 (45.0)	86 (52.4)	86 (48.0)		102 (54.3)

HCD: Historical Control Data (2015-2016; New Zealand White rabbits): maximal study fetal incidence (maximal study litter incidence) for the findings; -: none in HCD.

Table 9: Fetal skeletal variations

 

Dose-level (mg/kg/day)	0	100	300	1000		HCD
Number of litters	23	22	19	23		159
Number of fetuses	231	164	179	188		1558
Skull
Interparietal: incomplete ossification	0.9 (8.7)	2.4 (13.6)	5.0* (31.6)		4.8* (21.1)	4.1 (36.8)
Sternebra
Incomplete ossification of 1^stto 4^thsternebrae, F (L)	0.0 (0.0)	1.2 (9.1)	1.1 (10.5)		3.2** (17.4)	4.4 (26.3)
Extra sternebral ossification site	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)		0.5 (4.3)	1.2 (8.3)
Rib
Thickened rib(s), F (L)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)		1.6 (13.0)	1.0 (10.5)
Ossification point on 13^ththoracic vertebra	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)		0.5 (4.3)	0.6 (5.6)
Metacarpal bone
Incomplete ossification of 1^stmetacarpal, F (L)	5.6 (30.4)	3.7 (22.7)	6.7 (31.6)		11.7* (47.8)	8.4 (61.1)
Pelvis
Pubis: unossified, F (L)	0.0 (0.0)	0.0 (0.0)	1.1 (10.5)		0.0 (0.0)	0.5 (5.6)
Thoracic vertebrae
Dumbbell ossification of centrum	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)		0.5 (4.3)	-
Litters with skeletal variations, n (%)	23 (100.0)	22 (100.0)	19 (100.0)		23 (100.0)	159 (100)
Fetuses with skeletal variations, n (%)	202 (87.4)	139 (84.8)	151 (84.4)		170 (90.4)	1382 (88.7)

F: fetal incidence; L: litter incidence,n: number; Statistical significance (on number of fetuses affected not on the percentages presented in the table):*: p < 0.05, **: p < 0.01.

HCD: Historical Control Data (2015-2016; New Zealand White rabbits): maximal study fetal incidence (maximal study litter incidence) for the findings; -: none in HCD.

Table 10: Fetal skeletal malformations

Dose-level (mg/kg/day)	0	100	300	1000	HCD
Number of litters	23	22	19	23	159
Number of fetuses	231	164	179	188	1558
Head skull
Interparietal: split, F(L)	2.2 (21.7)	3.0 (22.7)	4.5 (31.6)	1.6 (13.0)	1.2 (11.1)
Interparietal: absent	3.9 (21.7)	1.2 (4.5)	3.4 (21.1)	0.5 (4.3)	3.4 (31.6)
Parietal: split	0.4 (4.3)	0.0 (0.0)	0.0 (0.0)	1.6 (13.0)	0.4 (4.2)
Skullcap: hole	0.9 (8.7)		0.6 (5.3)		0.3 (2.7)
Caudal vertebrae
Fused	0.4 (4.3)	0.6 (4.5)		0.5 (4.3)	0.9 (8.3)
Absent		0.6 (4.5)			0.4 (4.2)
Misaligned				0.5 (4.3)	-
Sternebrae
Fused	2.2 (13.0)	3.7 (18.2)	6.7* (26.3)	4.3 (17.4)	2.2 (20.8) [or 5.1 (31.3)^a]
Rib
Fused				0.5 (4.3)	0.9 (8.3)
Litters with skeletal malformations, n (%)	10 (43.5)	8 (36.4)	12 (63.2)	10 (43.5)	46 (28.9)
Fetuses with skeletal malformations, n (%)	20 (8.7)	13 (7.9)	27 (15.1)	17 (9.0)	56 (3.6)

F: fetal incidence; L: litter incidence;n: number; Statistical significance (on number of fetuses affected not on the percentages presented in the table):*: p < 0.05.

HCD: Historical Control Data (2015-2016; New Zealand White rabbits): maximal study fetal incidence (maximal study litter incidence) for the findings; -: none in HCD.

^a: HCD from 2013-2014.

Conclusions:

On the basis of the experimental conditions and results obtained in this study, the dose of 1000 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) for maternal parameters and for embryo-fetal development, based on the non-adverse effects on dam body weight change and food consumption and on non-adverse increased incidences of the fetal gall bladder and skeletal variations at this dose.

Executive summary:

The objective of this GLP study was to evaluate the potential toxic effects of the test item on the pregnant female and on embryonic and fetal development, following daily oral administration (gavage), to pregnant female rabbits from implantation to the day prior the scheduled hysterectomy [Days 6 to 28 post-coitum (p.c.) inclusive].

 

Methods

Three groups of 24 time-mated female New Zealand White rabbits received the test item by oral route (gavage) at doses of 100, 300 and 1000 mg/kg/day once daily from Day 6 post-coitum (p.c.) until Day 28 p.c. inclusive. One additional group of 24 time-mated females received the vehicle [methylcellulose (1%) in drinking water treated by reverse osmosis] under the same experimental conditions and acted as a control group. A constant dose-volume of 3 mL/kg/day was used.

Formulation concentrations were checked twice (at the beginning and at the end of the study).

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded every 1 to 5 days from their arrival.

On Day 29 p.c., females were euthanized and submitted to a macroscopic post-mortem examination of the principal thoracic and abdominal organs. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, uterine scars, early and late resorptions, and live and dead fetuses were recorded. The fetuses were sexed, weighed and examined for external, soft tissue and/or skeletal (bones and cartilages) abnormalities. Placentas were grossly observed and weighed.

 

Results

Test item concentrations in the dose formulations analyzed during the study were within an acceptable range of variations when compared to the nominal values (± 15% accepted) and there was no test item in the control dose formulations.

On Day 29 p.c., there were 23/24, 22/24, 19/23 and 23/23 pregnant females with live fetuses in control, 100, 300 and 1000 mg/kg/day groups, respectively.

At 1000 mg/kg/day, there was a premature euthanasia following abortion; clinical signs, body weight loss and poor food consumption were observed beforehand. A relationship between the test item treatment and the abortion could not be excluded.

There were no other clinical signs or necropsy findings considered to be test item-related.

There were no test item effects on mean body weight and mean food consumption at 100 and 300 mg/kg/day. At 1000 mg/kg/day, there were slight mean body weight loss on Days 6 to p.c.(-16 g vs. +32 g in controls, p<0.05) and on Days 24 to 29 p.c.(-18 g vs.+31 g) and a low mean body weight gain on Days 9 to 12 p.c.(+23 g vs.+71 g, p<0.05). Concomitant low mean food consumption was noted (-21 to -27% on Days 6 to 15 p.c. vs. controls, p<0.05 or p<0.001). These effects were considered to be test item-related and non-adverse (no toxicological impact on mean body weight, no strong differences from controls).

There were no test item-related effects on mean uterus weight, mean carcass weight and mean net body weight change in any groups.

 

There were no test item-related effects on mean hysterectomy data and mean fetal sex ratio in any groups.

There were no toxicologically significant differences in mean fetal and placental weights data between groups in any groups.

There were no test item-related external variations and no external and visceral malformations in litters in any groups. A test item relationship with increased skeletal variation incidences at 300 and 1000 mg/kg/day (mainly incomplete ossification of 1^st to 4^thsternebrae, interparietal and 1^stmetacarpal bone; thickened ribs) were considered uncertain but non-adverse.

At 1000 mg/kg/day when compared with controls, there were higher litter and fetal incidences of variations on the gall bladder (8 fetuses among 6 litters affected vs. 2 fetuses among 2 litters in controls). These findings were considered to be test item-related and non-adverse.

At 300 and 1000 mg/kg/day, a test item treatment effect on slightly increased incidences of split interparietal (4.5% of fetuses and 31.6% of litters affected at 300 mg/kg/day vs. 2.2% and 21.7% in controls, respectively) or parietal (1.6% of fetuses and 13.0% of litters affected vs .0.4% and 4.3% in controls, respectively) could not be excluded but considered as non-adverse since the differences from control incidences were low (no statistical differences), since the findings were of low incidences and do not impact the global shape of the skull and since they are probably due to ossification delay.

 

Conclusion

On the basis of the experimental conditions and results obtained in this study, the dose of 1000 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) for maternal parameters and forembryo-fetal development, based on the non-adverse effects on dam body weight change and food consumption and on non-adverse increased incidences of fetal gall bladder and skeletal variations at this dose.

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:

other: rat and rabbit

Quality of whole database:

Both studies performed on rats and rabbits are reliable with a klimisch score of 1.

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

Key study : Developmental toxicity study in rat (Braun 2015) :

The oral (gavage) prenatal developmental toxicity of the test item 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid (CAS 55818-57-0, DGEBADA) to Wistar rats was investigated in a GLP-compliant dose-effect study according to the OECD TG 414 (2001).

The purpose of this study was to detect effects on the pregnant rat and development of the embryo and foetus consequent to exposure of the female to the test item from day 6 post coitum (implantation) to day 20 post coitum (the day prior to caesarean section). Four groups of 24 mated females per group were treated by gavage with DGEBADA once daily at nominal dose levels of 0 (control group), 100, 300 and 1000 mg/kg body weight/day (named groups 1 to 4, respectively). A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (PEG 300). All females were sacrificed on day 21 post coitum and the foetuses were removed by caesarean section.

Regarding the maternal data, no test item-related premature deaths were recorded. One female in group 3 was sacrificed for ethical reasons unrelated to the treatment with the test item. No test item-related clinical symptoms or signs, no changes in the food consumption and no effects on mean absolute body weights and body weight gain were observed at any dose during the study. The relevant reproduction data (post-implantation loss and number of foetuses per dam) were not affected by treatment with the test item and no test item-related macroscopic findings were observed at any dose level during macroscopic examination.

The foetal examinations revealed in group 4 placenta weights on a litter basis being increased by 23 and by 24 % on an individual basis. This increase was considered to be test item-related. Differences noted in groups 2 and 3 were unrelated to dose and therefore considered to be incidental. No test findings were observed during external examination of the foetuses. No test item-related effects on sex ratio and mean body weights of the foetuses were noted in any group. No test item-related abnormalities and variations were noted during external and fresh visceral examinations of foetuses and no findings were noted during skeletal examination of the foetuses. A small number of incomplete or non-ossified bones and/or supernumerary ribs remained within the ranges of the respective historical control values and were therefore considered to be of no toxicological relevance. There were no additional cartilage finding in any foetus that was considered to be related to the treatment with the test item.

In conclusion based on these results, the NOEL (No Observed Effect Level) and NOAEL (No Observed Adverse Effect Level) for maternal toxicity was considered to be above 1000 mg/kg bw/day. Placenta weight differences seen in group 4 were considered to be test item-related changes; the NOEL for prenatal development was therefore considered to be 300 mg/kg/day. The NOAEL for prenatal developmental toxicity was defined as 1000 mg/kg bw/day.

Key study : Developmental toxicity study in rabbit (Bentz 2017) :

Three groups of 24 time-mated female New Zealand White rabbits received the test item by oral route (gavage) at doses of 100, 300 and 1000 mg/kg/day once daily from Day 6 post-coitum (p.c.) until Day 28p.c.inclusive. One additional group of 24 time-mated females received the vehicle [methylcellulose (1%) in drinking water treated by reverse osmosis] under the same experimental conditions and acted as a control group. A constant dose-volume of 3 mL/kg/day was used.

On Day 29 p.c.,there were 23/24, 22/24, 19/23 and 23/23 pregnant females with live fetuses in control, 100, 300 and 1000 mg/kg/day groups, respectively.

At 1000 mg/kg/day, there was a premature euthanasia following abortion; clinical signs, body weight loss and poor food consumption were observed beforehand. A relationship between the test item treatment and the abortion could not be excluded.

There were no other clinical signs or necropsy findings considered to be test item-related.

There were no test item effects on mean body weight and mean food consumption at 100 and 300 mg/kg/day. At 1000 mg/kg/day, there were slight mean body weight loss on Days 6 top.c.(-16 g vs. +32 g in controls, p<0.05) and on Days 24 to 29p.c.(-18 gvs.+31 g) and a low mean body weight gain on Days 9 to 12p.c.(+23 gvs.+71 g, p<0.05). Concomitant low mean food consumption was noted (-21 to -27% on Days 6 to 15 p.c.vs.controls, p<0.05 or p<0.001). These effects were considered to be test item-related and non-adverse (no toxicological impact on mean body weight, no strong differences from controls).

There were no test item-related effects on mean uterus weight, mean carcass weight and mean net body weight change in any groups.

There were no test item-related effects on mean hysterectomy data and mean fetal sex ratio in any groups.

There were no toxicologically significant differences in mean fetal and placental weights data between groups in any groups.

There were no test item-related external variations and no external and visceral malformations in litters in any groups. A test item relationship with increased skeletal variation incidences at 300 and 1000 mg/kg/day (mainly incomplete ossification of 1st to 4th sternebrae, interparietal and 1st metacarpal bone; thickened ribs) were considered uncertain but non-adverse.

At 1000 mg/kg/day when compared with controls, there were higher litter and fetal incidences of variations on the gall bladder (8 fetuses among 6 litters affectedvs.2 fetuses among 2 litters in controls). These findings were considered to be test item-related and non-adverse.

At 300 and 1000 mg/kg/day, a test item treatment effect on slightly increased incidences of split interparietal (4.5% of fetuses and 31.6% of litters affected at 300 mg/kg/dayvs.2.2% and 21.7% in controls, respectively) or parietal (1.6% of fetuses and 13.0% of litters affectedvs .0.4% and 4.3% in controls, respectively) could not be excluded but considered as non-adverse since the differences from control incidences were low (no statistical differences), since the findings were of low incidences and do not impact the global shape of the skull and since they are probably due to ossification delay.

On the basis of the experimental conditions and results obtained in this study, the dose of 1000 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) for maternal parameters and forembryo-fetal development, based on the non-adverse effects on dam body weight change and food consumption and on non-adverse increased incidences of fetal gall bladder and skeletal variations at this dose.

Toxicity to reproduction: other studies

Additional information

The adverse effects observed in the 13 -week repeated toxicity study at 100, 300 and 1000 mg/kg were not reproducible in the extended one-generation reproduction study in which males were exposed 12 weeks and females 13 -16 weeks to the doses of 40, 100 and 200 mg/kg/day. The same species and the same type of vehicle were use in both studies.

In the 13 -week repeated toxicity study, reduced locomotor activity (in males at all dose levels and in females treated at 1000 mg/kg bw/day), reduced sperm motility at all dose levels, lower mean absolute and relative prostate weights at 100, 300 and 1000 mg/kg/day and multifocal cell hypertrophy observed in the pars distalis of the pituitary gland of males treated at 1000 mg/kg/ day. The pituitary findings and the decreased prostate/seminal vesicles weights may represent an indicator of mild disruption of testosterone production/levels. So, adverse effects on male fertility were supposed.

This type of effects was not observed in the previous reproduction screening (OECD 422). This could be explained by the low exposure of male before mating (2-week only) instead of the 10 -week premating exposure in the OECD 443 study.

That is why the results obtained in the OECD 422 were not used for selecting dose of the OECD 443.

The 90 -day study alone was used for selecting the doses of the extended one-generation reproduction study with the objective to have litters at all tested doses :

-         The low dose of 40 mg/kg/day. No adverse male fertility was expected at this dose, so complete analyses of litter could be done.

-         The intermediate dose at 100 mg/kg/day, dose already used in the 90 day study to compare the previous results obtained.

-         The high dose of 200 mg/kg, because the dose of 300 mg/kg/day is considered to be too high based on the 90-day study.

At this end, no adverse effect on locomotor activity, on prostate weight, on semiology and on pituitary were observed in the OECD 443.

The results obtained in the OECD 443 were considered to be more relevant due to the number of animal used (25 animals/sex/dose instead of 10 animals/sex/dose in the OECD 422).

The effects observed in the 90 -day study were considered to be not-treatment related and due to chance findings.

Pups and parental toxicities were observed at 100 and 200 mg/gk/day in the OECD 443 even if these effects were not considered to be adverse.

Justification for classification or non-classification

Based on the absence of adverse effect on the reproduction parameters in rats and on developmental toxicity in rats and rabbits, no classification of DGEBADA is required for reproduction according to the CLP Regulation (EC) N°1272-2008.