2,2-dimethyl-1,3-dioxolan-4-ylmethanol

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test performed in rats, the dose-level of 1000 mg/kg/day was considered to be the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 13 March 2013 to 19 November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD Guideline 422 without any major deviation

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificat n° 2012/96 ; 10 January 2013

Limit test:

no

Specific details on test material used for the study:

- Analytical purity: 99.9%
- Composition of test material, percentage of components: 99.9% 2,2-Dimethyl-1,3-dioxolane-4-methanol; water: 0.02%; Acidity (Acetic acid): 0.0021%
- Lot/batch No.: BR12K853
- Expiration date of the lot/batch: 05 November 2013
- Storage condition of test material: at room temperature

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Italia, Calco, Italy.
- Age at study initiation: Males: 10 weeks; Females: 9 weeks
- Weight at study initiation: Males: 388 g (331 to 440 g); females: 230 g (194 to 264 g)
- Housing: Individually, except during pairing and lactation, in polycarbonate cages (Tecniplast 2154, 940 cm²) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were provided to females and their litter as nesting material.
- Diet: SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Drinking water filtered with a 0.22 µm filter, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 50 ± 20%
- Air changes: 12 air changes per hour of filtered, non-recycled air
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From 19 March 2013 to 10 May 2013.

Route of administration:

oral: gavage

Vehicle:

physiological saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle. The test item was dissolved with the required quantity of vehicle to give the required concentrations of 50, 100 and 200 mg/mL. The formulations were prepared for up to 9 days, divided into daily aliquots and stored at room temperature and protected from light. For each dosing, daily aliquots were delivered to the study room protected from light

VEHICLE
- Concentration in vehicle: 50, 100 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw.

Details on mating procedure:

- M/F ratio per cage: 1: 1
- Length of cohabitation: Overnight
- Proof of pregnancy: presence of a vaginal plug or for sperm in a vaginal lavage referred to as Day 0 p.c..
- Each female was placed with the same male until mating occurs. The pre-coital time was calculated for each female. The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until the females were mated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The concentrations of the dose formulations were checked in study weeks 1, 3 and 6 by a validated analytical method (CiToxLAB France/Study No. 39832 VAA).
- Acceptance criterion: measured concentration = nominal concentration ± 10%
- The pH of the obtained solutions used throughout the study was measured by means of a pH-meter.
- Results: The test item concentrations in the administered dose formulations analyzed in weeks 1, 3 and 6 were within an acceptable range of variation (i.e. -2% to +8%) when compared to the nominal values (± 10%); pH was close to neutrality.

Duration of treatment / exposure:

- Males: 2 weeks before pairing, during the pairing period (up to 5 days), until sacrifice (5 weeks in total).
- Females: 2 weeks before pairing, during the pairing period (up to 5 days), during gestation, during lactation until day 5 post partum (p.p.) inclusive.
- Day 1 corresponds to the first day of the treatment period.

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
250, 500 or 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose-levels were selected in agreement with the Sponsor, following the results of a previous 2-week toxicity study performed with the same species at the dose-levels of 250, 500 or 1000 mg/kg bw/day (Study No. 39834 TSR). In this study, no toxicologically relevant findings, but a slight decrease in body weight in females given 1000 mg/kg bw/day when compared to controls were observed.
- Rationale for animal assignment: during the acclimation period, the required number of animals (40 males and 40 females) was selected according to body weight and clinical condition. The animals were allocated to groups (by sex) according to computerized stratification procedure based on body weight, so that the average body weight of each group was similar.

Positive control:

Not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS (PARENTAL ANIMALS): Yes
- Time schedule:
Viability / Mortality: Once a day before the treatment period and twice a day during the treatment period. A female of group 4 showing signs of poor clinical condition (immobilized hindlimbs), was humanely sacrificed on day 4 p.c., and was subjected to a macroscopic post-mortem examination.
Clinical Signs: Once daily, during acclimatization and up to day of necropsy. As a male from the control group displayed liquid feces on day 22, decision was taken to weigh and to examine the animal closely on day 23.

DETAILED CLINICAL OBSERVATIONS (PARENTAL ANIMALS): Yes
- Time schedule: Detailed clinical examinations were performed on all animals once before the beginning of the treatment period and then once a week until the end of the study. Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded.

NEUROBEHAVIOURAL EXAMINATION (PARENTAL ANIMALS): Yes
- Time schedule: The first five males and the first five females to be sacrificed on day 6 p.p. from each group were evaluated once at the end of the treatment period. For females, this was performed on day 5 p.p. after sacrifice of the pups.
- Animals were observed for the following:
Cage-side observations: "touch escape" or ease of removal from the cage.
Hand-held observations: fur appearance, salivation, lacrimation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis).
Standard arena (2-minute recording) observations: grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper- activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.
Reactivity to manipulation and to different stimuli: The following parameter measurements, reflexes and responses were recorded: touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, at the end of observation: rectal temperature.
Motor activity: Finally, motor activity was measured once by automated infra-red sensor equipment over a 60-minute period.


OPHTHALMOSCOPIC EXAMINATION: No data

BODY WEIGHT (PARENTAL ANIMALS): Yes
- Time schedule for examinations: The body weight of each male was recorded on the first day of treatment (day 1), then once a week until sacrifice. The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mated, on days 0, 7, 14 and 20 post-coitum (p.c.) and days 1 and 5 p.p..
- The body weight of each animal sacrificed as scheduled after the end of the pairing period for males or on day 6 p.p. for females was recorded before sacrifice.

FOOD CONSUMPTION (PARENTAL ANIMALS): Yes
- Time schedule for examinations: The quantity of food consumed by each male was measured once a week, over a 7-day period, from the first day of treatment until the start of the pairing period. The quantity of food consumed by each female was measured once a week, over a 7-day period, from the first day of treatment until the start of the pairing period, during pregnancy for the intervals days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval days 1-5 p.p.

HAEMATOLOGY (PARENTAL ANIMALS): Yes
- Time schedule for collection of blood: from the first five males and the first five females to be sacrificed on day 6 p.p. from each group on the day of sacrifice.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Blood samples were taken from the orbital sinus of the animals, under light isoflurane anesthesia, into appropriate tubes.
- Animals fasted: Yes; an overnight period of at least 14 hours
- How many animals: five males and five females
- Parameters checked: Erythrocytes, Mean cell volume, Packed cell volume, Hemoglobin, Mean cell hemoglobin Concentration, Mean cell hemoglobin, Thrombocytes, Leucocytes, Differential white cell count with cell morphology (neutrophils, eosinophils, basophils, lymphocytes and large unstained cells, monocytes), Reticulocytes, Prothrombin time, Fibrinogen, Activated partial thromboplastin time.

CLINICAL CHEMISTRY (PARENTAL ANIMALS): Yes
- Time schedule for collection of blood: from the first five males and the first five females to be sacrificed on day 6 p.p. from each group on the day of sacrifice.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Blood samples were taken from the orbital sinus of the animals, under light isoflurane anesthesia, into appropriate tubes.
- Animals fasted: Yes; an overnight period of at least 14 hours
- How many animals: five males and five females
- Parameters checked: Sodium, Potassium, Chloride , Calcium, Inorganic phosphorus, Glucose, Urea, Creatinine, Bilirubin (total), Cholesterol (total), Triglycerides, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Protein (total), Albumin, Albumin/Globulin ratio and Bile acids

URINALYSIS: No data

PREGNANCY AND PARTURITION:
- Females were allowed to litter normally and rear their progeny until day 5 p.p.. Any sign of a difficult or prolonged parturition was recorded. The morning when the parturition was completed was designated day 1 p.p.. The length of gestation was calculated.

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until the females were mated.

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight, epididymis weight, detailed histological examination of the testes and epididymides with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Litters were examined for litter size, sex of each pup and any gross anomalies.
- The litters were observed daily in order to note the number of live, dead and cannibalized pups.
- The pups were observed daily for clinical signs, abnormal behavior and external abnormalities.
- The body weight of each pup was recorded on days 1 and 5 p.p..

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

Postmortem examinations (parental animals):

SACRIFICE (PARENTAL ANIMALS)
- On completion of the treatment period, after at least 14 hours fasting, all surviving F0 animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination: males: after the end of the pairing period (5 weeks of treatment in total); females: on day 6 p.p..
- One female was prematurely sacrificed during the gestation period. This female was deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination.

GROSS PATHOLOGY: Yes
- All parent animals were submitted to complete macroscopic post-mortem examination, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death. This included examination of the external surfaces, all orifices, the cranial
cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.
- For prematurely sacrificed animals the pregnancy status was determined. The presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique. Implantation sites and corpora lutea were counted and recorded for females sacrificed as scheduled on day 6 p.p..

ORGAN WEIGHTS
Body weight of each animal was recorded before sacrifice and the organs specified in the table 7.8.1/2 were weighed. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

HISTOPATHOLOGY: Yes
- Samples of the tissues specified in the table 7.8.1/2 were preserved in 10% buffered formalin (except for the testes and epididymides which were fixed in modified Davidson's fixative).
- All tissues required for microscopic examination were trimmed, embedded in paraffin wax and sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except testes and epididymides which were stained with hematoxylin/PAS). Microscopic examination was performed on all tissues listed in the table 7.8.1/2 from the first five sacrificed as scheduled males and the first five females sacrificed on day 6 p.p. of the control and high-dose groups (groups 1 and 4) and from the prematurely sacrificed female, and on all macroscopic lesions of all groups. Based on the microscopic findings seen in the kidneys of high-dose males, a microscopic examination was performed on the kidneys from group 2 and group 3 males. Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Postmortem examinations (offspring):

SACRIFICE (LITTER):
- Surviving pups were sacrificed by an intraperitoneal injection of sodium pentobarbital on day 5 p.p..

GROSS NECROPSY (LITTER):
- A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all found dead pups and on all pups sacrificed on day 5 p.p.. Special attention was paid to the reproductive organs and to whether the pup has fed (e.g. presence of milk in the stomach). No tissues were preserved.

Statistics:

- Data are expressed as group mean values ± standard deviation (body weight, body weight change, food consumption, number of corpora lutea, number of implantation sites, number of pups and pup body weight, gestation length) or as proportions (pre-implantation loss, post-implantation loss, pup observations, mating index, fertility index, gestation index, live birth index, viability index). Whenever appropriate, the experimental unit of comparison was the litter. Data of the non-pregnant female are not included in group mean calculations such as body weight, body weight change, food consumption.
- Body weight, food consumption and reproductive data are compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions).
- Hematology and blood biochemistry data are compared by various tests according to a decision tree: Dunnett-test, Dunn test or Mann-Whitney / Wilcoxon test (according to variance homogeneity between groups) were applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups. Mann-Whitney / Wilcoxon test or Dunn test were applied when the data could not be assumed to follow a normal distribution.
- PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01) by Dunn test if >2 groups or Wilcoxon test if 2 groups and Dunnett test if >2 groups or t-test if 2 groups, based on normal distribution and homogeneity of variance.

Reproductive indices:

Pre-implantation loss = (Number of corpora lutea - Number of implantation sites)/ Number of corpora lutea x 100
Post-implantation loss (calculated manually) = (Number of implantation sites - Number of live pups)/ Number of implantation sites x 100
Mating index = Number of mated animals / Number of paired animals x 100
Fertility index = Number of pregnant female partners / Number of mated pairs x 100
Gestation index = Number of females with live born pups / Number of pregnant females x 100

Offspring viability indices:

Live birth index = Number of live born pups / Number of delivered pups x 100
Viability index = Number of surviving pups on day 4 p.p. / Number of live born pups x 100

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- There were no deaths considered as test item-related. One female given 1000 mg/kg/day was prematurely sacrificed on day 4 p.c. on ethical grounds as this animal displayed hindlimb immobilized before sacrifice. No necropsy findings were noted. In view of the duration of the treatment, and absence of pre-neoplastic or neoplastic hematopoietic lesions in other test item-treated animals, this isolated finding was considered to be fortuitous. There were no other unscheduled deaths during the study.
- There were no test item treatment-related clinical signs.

BODY WEIGHT AND WEIGHT GAIN (PARENTAL ANIMALS)
- There were no test item treatment-related effects on mean body weight or mean body weight gain.
- When compared with the control group, a slightly lower mean body weight gain was observed in females treated at 1000 mg/kg/day during the whole pre-mating period (+30 g and +18 g between Days 1 – 15, respectively). When compared with the control group, the opposite trend was noted in all test item-treated males with a statistical significance over the last 3 weeks at 250 and 1000 mg/kg/day (+31 g, +50 g and +49 g between Days 15 – 36, respectively). Since these differences were not dose-related and of opposite tendency and since these differences had no impact on the final body weights, they were considered not to be relevant.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- When compared with the mean control values, females treated at 500 and 1000 mg/kg/day had statistically significantly lower food consumption (23 g/rat/day, 20 g/rat/day and 21 g/rat/day between Days 1 – 8 of Pre-mating, respectively). As this effect was minimal, not dose-related and limited to the first week of the pre-mating period, it was considered to be of no toxicological importance. There were no effects on mean food consumption at these dose-levels in males.
- The test item did not affect the food consumption at any of the dose-levels employed.

NEUROBEHAVIOURAL EXAMINATION (PARENTAL ANIMALS)
- No relevant findings were noted during functional observational battery testing in test item-treated groups when compared with the control groups.

LABORATORY INVESTIGATIONS (PARENTAL ANIMALS)
- There were no test item-related findings on hematology parameters. Lower mean values of Mean Cell Hemoglobin, prothrombin and activated partial thromboplastin times recorded in males given the high dose-level were considered to be of no toxicological importance as they were minimal, with no biological significance and/or isolated variations.
- When compared with the mean control values, the cholesterol concentration of males treated at 1000 mg/kg/day was 77% higher (2.3 vs. 1.3 mmol/L, p<0.01). In absence of treatment-related microscopic findings in the liver, these variations were considered to be of minor toxicological importance.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- There were no evident signs of disturbances of the estrus cycle. There were no test item-related effects on mating and fertility data.
- At 1000 mg/kg/day, the higher pre-implantation loss (11.2% instead of 5.2 % in control group) was considered to be fortuitous in origin and not considered to be related to the test item administration since the number of pups per litter was unaffected. There were no effects on the mean numbers of corpora lutea, implantations or pups at any dose-level, nor on the duration of gestation or the extent of post-implantation losses.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- Test item-related changes were observed for the liver in males. There were no test item-related organ weight changes in females. When compared with controls, there was a slight increase in the mean absolute and relative liver weights in males given the test item at 1000 mg/kg/day (+31% and +26%, respectively), reaching statistical significance for the relative weight only. A minimal trend was also present at 500 mg/kg/day (+22% and + 18%, respectively), but the differences were not statistically significant. These variations were considered to be related to the test item but were considered not to be adverse in view of the slight magnitude of the changes.
- The mean absolute and relative spleen weights were slightly higher in males given the test item at 1000 mg/kg/day compared with controls (up to +29%). This correlated at microscopic examination with increased congestion in two animals, and was considered to be an agonal phenomenon related to euthanasia with pentobarbital.
- Other occasional organ weight changes were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, and/or were not dose-related in magnitude.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- In the vagina of the female given 1000 mg/kg/day prematurely sacrificed on day 4 p.c., there was a slight subacute inflammation, and moderate mucification of the mucosal epithelium. Low development of the corpora lutea in the ovaries and slight glandular atrophy in the stomach (fundus) may have been secondary to stress and poor clinical condition of this animal. None of these findings were attributed to the test item.
- There were no macroscopic findings attributed to the test item administration. The thymus was reduced in size in a single female in each of the 500 and 1000 mg/kg/day dose-groups.
This correlated at microscopic examination with lymphoid atrophy. However lymphoid atrophy was recorded with similar incidence in controls and high-dose females. These macroscopic findings were therefore considered to be incidental and unrelated to the test item administration.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- There were no effects on the genital organs examined in any groups. A careful qualitative histopathological examination of the testes was performed. There were no effects on the germ cells or interstitial (Leydig) cells.
- Test item-related microscopic findings occurred in the kidneys from males. There were no test item-related findings in females. In the kidneys, tubular hyaline droplets were seen with increased incidence and severity in males given the test item at 1000 mg/kg/day compared with controls. This was characterized by the presence of dense eosinophilic droplets in proximal tubular epithelium. These hyaline droplets, occasionally seen in untreated male rats, are consistent with α2u globulin. Although human excrete proteins of a similar nature, they are found in only trace amounts and therefore this finding is considered to be non-relevant for human. There were no significant findings at 250 and 500 mg/kg/day.
- Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the Sprague-Dawley rat.

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOEL

Remarks:

(reproductive toxicity)

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed on mating, fertility and delivery data in any group

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

OFFSPRING LITTER SIZE AND VIABILITY
- There were no effects on pup viability and none of the observed clinical signs were considered to be test item-related (comparable incidences in control pups or not dose-related).
- There were no effects of treatment with the test item on the percentage of male pups at birth at any dose-levels.

OFFSPRING GROWTH AND DEVELOPMENT
- There were no effects of treatment with the test item on mean body weight and mean body weight change in pups.
- The macroscopic findings were considered not to be of toxicological importance as these findings can appear spontaneously and as they were no test item-related (no dose-relationship and slight incidences).

Dose descriptor:

NOEL

Remarks:

(developmental toxicity)

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed on pups by day 5 post partum (viability, clinical signs, sex ratio, macroscopic post-mortem findings)

Reproductive effects observed:

not specified

None

Conclusions:

Under the test condition, the dose-level of 1000 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) for parental and systemic toxicity (based on findings in the kidneys of males) and the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) and for toxic effects on progeny.

Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, 2,2-Dimethyl-1,3-dioxolane-4-methanol diluted in Phosphate Buffer Saline solution was administered daily by oral gavage to male and female Sprague-Dawley rats (10/sex/dose), for 2 weeks before mating, during mating and (for females) throughout gestation and until day 5 post-partum, at dose-levels of 250, 500 or 1000 mg/kg bw/day. A control group was treated with Phosphate Buffer Saline solution. During the study, data was recorded on mortality, clinical signs, behavioural assessments, body weight change, food consumption, haematology, blood chemistry, mating performance, fertility and gestation length. All animals were subjected to a gross necropsy examination, selected organs were weighed and histopathological evaluation of selected tissues was performed. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken.

 

No test item-related deaths or clinical signs were noted in any group and sex. No toxicologically relevant effects on mean body weight, mean food consumption, mean Functional Observation Battery and motor activity data and mean clinical pathology parameters in any group and sex. The increase in the absolute and relative liver weights recorded in males given 1000 mg/kg bw/day was considered not to be adverse in view of the slight magnitude of the changes and the absence of microscopic correlates. None of the macroscopic findings were attributed to the test item administration. At histopathology, tubular hyaline droplets in the kidneys (consistent with rat-specific alpha-2u-globulin) were seen with increased incidence and severity in males treated at 1000 mg/kg bw/day compared to controls (presence of dense eosinophilic droplets in proximal tubular epithelium). This finding was not observed at 250 or 500 mg/kg bw/day. These kidney effects were considered to be related to alpha-2u globulin nephropathy and of no relevance to humans. There were no toxicologically relevant effects on mean mating, fertility and delivery data in any group. There were no test item-related effects on pups by day 5 post partum (viability, clinical signs, sex ratio, macroscopic post-mortem findings).

 

Under the test condition, the dose-level of 1000 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) for parental and systemic toxicity (based on findings in the kidneys of males) and the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) and for toxic effects on progeny.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP study conducted according to OECD Guideline 422 without any major deviation (Klimisch score = 1).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, 2,2-Dimethyl-1,3-dioxolane-4-methanol diluted in Phosphate Buffer Saline solution was administered daily by oral gavage to male and female Sprague-Dawley rats (10/sex/dose), for 2 weeks before mating, during mating and (for females) throughout gestation and until day 5 post-partum, at dose-levels of 250, 500 or 1000 mg/kg bw/day. A control group was treated with Phosphate Buffer Saline solution.

No test item-related deaths or clinical signs were noted in any group and sex. No toxicologically relevant effects on mean body weight, mean food consumption, mean Functional Observation Battery and motor activity data and mean clinical pathology parameters in any group and sex. The increase in the absolute and relative liver weights recorded in males given 1000 mg/kg bw/day was considered not to be adverse in view of the slight magnitude of the changes and the absence of microscopic correlates. None of the macroscopic findings were attributed to the test item administration. At histopathology, tubular hyaline droplets in the kidneys (consistent with rat-specific alpha-2u-globulin) were seen with increased incidence and severity in males treated at 1000 mg/kg bw/day compared to controls (presence of dense eosinophilic droplets in proximal tubular epithelium). This finding was not observed at 250 or 500 mg/kg bw/day. These kidney effects were considered to be related to alpha-2u globulin nephropathy and of no relevance to humans. There were no toxicologically relevant effects on mean mating, fertility and delivery data in any group. There were no test item-related effects on pups by day 5 post partum (viability, clinical signs, sex ratio, macroscopic post-mortem findings).

Under the test condition, the dose-level of 1000 mg/kg/day was considered to be the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility).

Effects on developmental toxicity

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 01 FEB 2022 to 12 APR 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to the OECD TG 414 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Temperature set to maintain controlled room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability analysis performed in another study demonstrated that the test material is stable in the vehicle when prepared and stored under the same conditions at concentrations between 1 and 200 mg/mL for 9 days. Dose formulation homogeneity was also demonstrated by GC/FID analysis.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: not expected
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: not relevant

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: None

Species:

rabbit

Strain:

New Zealand White

Remarks:

Crl: KBL(NZW)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratoires France -ESD, Chatillon sur Chalaronne, France
- Age at study initiation: 4 to 5 months
- Weight at study initiation: 2950 to 4265 g
- Fasting period before study: no
- Housing: single housed in noryl cages. For psychological / environmental enrichment, animals were provided with dumbbell and hay except when interrupted by study procedures/activities. Music was also put on for the same purpose.
Females were gently handled every day from arrival in order to allow habituation to manipulation.
- Diet: Pelleted complete diet, batch No. 102494 (Diet Reference No. 3409, KLIBA) and compacted hay pellets, batch No. 6801103 (SSNIFF), sterilized by irradiation, ad libitum. In addition, in case of drastically reduced food consumption during the acclimation period (≤ 50 g/animal/day), carrots were distributed daily. It was considered that there were no known contaminants in the feed that interfered with the objectives of the study.
- Water: Municipal tap water filtered with a 0.22 μm filter, ad libitum. It was considered that there were no known contaminants in the water that interfered, with the outcome of the study.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 to 21
- Humidity (%): 30 to 70
- Air changes (per hr): 5 to 15 (filtered, non-recycled air)
- Photoperiod (hrs dark / hrs light): 8 / 16

IN-LIFE DATES: From: 02 MAR 2022 To: 12 APR 2022

Route of administration:

oral: gavage

Vehicle:

other: Phosphate Buffer Saline solution (PBS) pH 7.4 (1X)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dose formulations were prepared according to stability data and divided into aliquots where required to allow them to be dispensed on each dosing occasion.
Control item was administered as a solution and test item as a suspension.

VEHICLE
- Justification for use and choice of vehicle: Stability analyses performed previously in Study No. 39833 AHS demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
- Concentration in vehicle: 0, 20, 60 and 200 mg/mL
- Amount of vehicle: 5 mL/kg/day
- Lot/batch no.: 2276826 (expiry JUN 2023) and 2276826 (NOV 2023)
- Purity: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were analyzed by Gas Chromatography with FID detection (GC/FID) for the determination of AUGEO SL 191.
The dose formulations prepared in the first, second and last weeks of treatment were all found to be within the acceptance criteria (measured concentrations within ± 15% of the theoretical concentrations). No test item was found in the control dose formulation.
Dose formulation homogeneity was also demonstrated, as the Relative Standard Deviation (RSD) was found to be ≤ 10% for each tested group.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant. Females were mated by the supplier and delivered on GD0 and GD1.

Duration of treatment / exposure:

From GD6 to GD28, inclusive

Frequency of treatment:

Once daily at approximately the same time of the day

Duration of test:

From GD 6 to GD 28 inclusive

Dose / conc.:

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

Remarks:

Group No. 1 (control item)

Dose / conc.:

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

Remarks:

Group No. 2

Dose / conc.:

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

Remarks:

Group No. 3

Dose / conc.:

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

Remarks:

Group No. 4

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- 1. Results from a tolerability study of the test material in non-pregnant rabbits where two groups of three females were administered by oral gavage for two periods of 7 days (with at least a 7-day washout) and staggered start between dose levels and groups (Study No. 49529 TSL (Hauray et al., 2022)). In this study, there were no unscheduled death and no test item-related clinical signs. A minimal body weight loss was in females treated at 1000 mg/kg bw/day, after 7 days of treatment (- 0.86% on Day 22 vs. Day 15 value) associated with minimal lower food intake. There were no effects at 300 mg/kg/day.
- 2. Results from a dose-range finding study of the test material in the pregnant rabbits where three group of six females were administered by oral gavage from GD6 to 28 inclusive, at 100, 300 or 1000 mg/kg bw/day. An additional group of 6 females received the vehicle only [Phosphate Buffer Saline solution (PBS) pH 7.4 (1X)] and served as a control group (Study No. 49530 RSL; Spézia et al., 2022). In this study, there were no test item related clinical signs, no adverse effects on body weight/body weight change, no effects on food consumption and no test-item related findings at maternal necropsy or hysterectomy. At external fetal examination, there were no test-item related findings.
- Rationale for animal assignment: computerized randomization procedure based on body weight recorded on GD0 or GD1 and provided by the breeder, to ensure comparatively similar mean body weights of the groups.

Maternal examinations:

MORTALITY: Yes
At least twice dailya (at the beginning and end of working day) beginning upon arrival through termination.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily beginning upon arrival through termination.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: In addition to the daily cageside observation, animals were observed between 1 and 3 hours post-dosing. A full clinical examination was performed on GD2 and on each weighing day during dosing phase and on the day of euthanasia.

BODY WEIGHT: Yes
- Time schedule for examinations: on GD2, 5, 6, 9, 12, 15, 18, 21, 24, 27 and 29.

FOOD CONSUMPTION: Measured daily from GD2.

WATER CONSUMPTION: Visual inspection of the water bottles.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #29 (intravenous injection of sodium pentobarbital)
- Organs examined: abdominal and thoracic cavities including the uterus.

Ovaries and uterine content:

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

Fetal examinations:

- External examinations: Yes: all fetuses
- Soft tissue examinations: Yes: all live fetuses
- Skeletal examinations: Yes: all live fetuses
- Head examinations: Yes: half of the fetuses
- Anogenital distance of all live rodent pups: No

Statistics:

Body weight, food consumption and reproductive data:
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:

See details in "Any other information on materials and methods incl. tables".

Historical control data:

Historical control data - 2021 on Reproductive toxicity provided by Charles River.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Only a few findings [loud breathing, wound/scab(s) and/or soft feces] were observed on only a few occasions and/or with no-dose level relationship. In addition, they are common observations in animals dosed by gavage and maintained under the experimental conditions of this study.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No effects on mean body weight.
No statistically significant differences in mean body weight changes compared to controls.
At 1000 mg/kg bw/day, there was a minimal body weight loss (-9 g vs. +27 g in controls on GD 6-9) with a return toward control values from GD 9-12. A test item-related effect cannot be excluded, but taking into account the reversibility and the amplitude of the change, this finding was considered to be non-adverse.
At 300 and 100 mg/kg bw/day, there were no effects on mean body weight change.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, mean food consumption was low from the beginning of the treatment period (down to -31% vs. controls on GD 7-8 and GD 8-9 with p < 0.01 and 0.001, respectively) with a return towards control values from GD 22-23. Taking into account the amplitude of the differences and the duration of the effect, this finding was considered to be test-item related and adverse.
At 300 and 100 mg/kg bw/day, there were no effects on mean food consumption.

Food efficiency:

not examined

Description (incidence and severity):

Not relevant.

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

not examined

Description (incidence and severity):

Not relevant.

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related effects on mean gravid uterus weight, mean carcass weight or mean net body weight change.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test-item related findings at the macroscopic examination.
No statistically significant differences in gross pathology findings compared to controls. Only a few findings (thymus/gall bladder discoloration, stomach with discolored area and/or periovarian serous cysts) were observed on only a few occasions and/or with no dose level relationship. In addition, they are common observations in the female New-Zealand white rabbit of this age and physiological condition.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Other effects:

no effects observed

Details on maternal toxic effects:

There were no effects on hysterectomy data (mean pre-/post-implantation losses and mean number of live fetuses).

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not specified

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

Description (incidence and severity):

not relevant

External malformations:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant differences compared to the controls.

Variations:
At 1000 mg/kg bw/day, one litter had 2 fetuses (4508-06 and 4508-12) with protruding tongue (variation) and open eyes (malformation). In addition, fetus 4508-12 had a local edema and an enlarged abdomen (variations).
At 300 and 100 mg/kg bw/day, there were no variations at external examination of the fetuses.

Malformations:
At 1000 mg/kg/day, three fetuses from 2 litters had a malformation that was not observed in controls:
• fetuses 4508-06 and 4508-12 with open eyes (bilateral),
• fetus 4522-10 with omphalocele (intestine protruding through the abdominal wall and
covered by a thin transparent membrane).
At 300 and 100 mg/kg bw/day, there were no malformations at external examination of the fetuses.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant differences compared to controls.

At 1000 mg/kg bw/day, absences of some cartilages were noted in fetus(es) with hemivertebra(e) (and hence absence of ribs).
From 100 mg/kg bw/day, the cartilage of hyoid was present, but not ossified.

Variations:
At 1000 mg/kg bw/day, when compared to the upper limit of the Historical Control Data, there were increased litter incidences of thoracic vertebra(e) with unossified arch(es) (4.8% vs. 0.0% in HCD) and unossified rib(s) (4.8% vs. 0.0 in HCD). These finding were considered to be test item-related.
From 100 mg/kg bw/day and when compared to controls, there was a dose-related increase in the incidence of unossified 5th sternebra. While the incidences remained close to upper limit of the HCD, a test-item relationship cannot be excluded.
All these findings were considered to represent delays in ossification (non-adverse test item effects).
All other fetal skeletal variations not presented in the above table were of isolated fetal and/or litter incidences, were not dose-related, and/or were of similar/lower incidence when compared to controls or Historical Control Data.

Malformations:
Fused sternebra and, supernumerary or absent pre-sacral vertebrae are not presented in the above table:
• fused sternebra(e) are no longer considered to be malformations, but normal stages in the development of a sternebra,
• supernumerary pre-sacral vertebra is described as supernumerary lumbar vertebra,
• absence of pre-sacral vertebra is described as absent lumbar vertebra.
At 1000 mg/kg bw/day, three fetuses from 2 different litters had at least one malformation at skeletal
examination (ossified bones):
• fetus 4502-04 with supernumerary lumbar vertebra (also described as supernumerary pre-sacral vertebra),
• fetus 4503-01 with fused thoracic vertebra(e),
• fetus 4503-02 with fused thoracic vertebra(e) and absent thoracic hemivertebra(e).
At 300 mg/kg/day, 2 litters had a fetus with a skeletal malformation (ossified bones):
• fetus 3517-05 with absent thoracic hemivertebra(e),
• fetus 3514-02 with absent lumbar vertebra (also described as the absence of pre-sacral vertebra).
At 100 mg/kg bw/day, 2 litters had a fetus with a skeletal malformation (ossified bones):
• fetus 2512-02 with branched rib(s),
• fetus 2507-07 with absent lumbar vertebra (also described as the absence of pre-sacral vertebra).
In controls, two fetuses from 2 different litters had a malformation at skeletal examination (ossified bones):
• fetus 1504-05 with split nasal bone,
• fetus 1514-04 with absent lumbar vertebra (also described as absence of pre-sacral vertebra).

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant differences compared to controls.

Variations:
At 1000 mg/kg bw/day, one litter had 2 fetuses (4507-06 and 4507-09) with liquid content in the cranial cavity (variation) associated with a marked dilated cerebral ventricle (malformation, see following section).
At 300 and 100 mg/kg bw/day, there were no test-item related variations at fetal soft tissue examinations.
The few other soft tissue variations are common observations in this strain of rabbit and the litter incidences were not dose-related and below or close to the upper limit of the Historical Control Data. Therefore, any relationship to the test-item was considered to be unlikely.

Malformations:
At 1000 mg/kg bw/day, there were 4 fetuses from 3 different litters with at least one soft-tissue malformation:
• fetuses 4507-06 and 4507-10 with bilateral microphthalmia (small eyes) and marked dilated cerebral ventricles,
• fetuses 4507-07 and 4508-06 with bilateral microphthalmia,
• fetus 4507-09 with marked dilated cerebral ventricle/short right ureter/dilated left
ureter/malpositioned kidney,
• fetus 4508-12 with a dilated aortic arch,
• fetus 4510-08 with a core trioculare (three-chambered heart, common atrium) and a dilated aortic arch.
At 300 mg/kg bw/day, one litter had a malformed fetus:
• fetus 3502-01 with a short right ureter and a malpositioned kidney.
At 100 mg/kg/day, there were no malformations at fetal soft tissue examinations. In controls, 3 litters had a malformed fetus:
• fetus 1509-02 with a core trioculare and a dilated aortic arch;
• fetuses 1518-08 and 1521-04 with a right malpositioned kidney and a short ureter.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

external: eye

skeletal: vertebra

other: brain

Description (incidence and severity):

11 malformed fetuses at 1000 mg/kg bw/day (6 abnormal litters; 1 to 4 malformed fetuses per litter)

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

no

MATERNAL TOXICITY

 

Pregnancy Status

Dose level (mg/kg bw/day)	0	100	300	1000
Number of time-mated females	22	22	22	22
Pregnant females	19	21	21	21
Females with live fetuses at termination	19	21	21	21
Non-pregnant females	3	1	1	1

 

Clinical signs

Dose level (mg/kg bw/day)	0	100	300	1000
Loud breathing	1 (from GD 25)
Wound (neck)			1 (from GD 27)
Scab(s) (interscapular region, ears, neck)			1 (GD 1-29)	2 (from GD 1)
Soft feces				1 (GD 10)

(): in brackets, period of occurrence.

 

Mean body weight (g)

Dose level (mg/kg bw/day)	0	100	300	1000
Body weight (g)
GD 2 (pre-treatment)	3466	3435	3459	3468
		(-1)	(0)	(0)
GD 5 (pre-treatment)	3487	3464	3537	3506
		(-1)	(+1)	(+1)
GD 6	3504	3468	3562	3530
		(-1)	(+2)	(+1)
GD 9	3531	3511	3601	3521
		(-1)	(+2)	(0)
GD 12	3578	3563	3654	3555
		(0)	(+2)	(-1)
GD 15	3650	3645	3729	3640
		(0)	(+2)	(0)
GD 18	3677	3655	3769	3665
		(-1)	(+3)	(0)
GD 21	3699	3698	3796	3709
		(0)	(+3)	(0)
GD 24	3741	3734	3843	3755
		(0)	(+3)	(0)
GD 27	3794	3788	3905	3812
		(0)	(+3)	(0)
GD 29	3868	3855	3967	3892
		(0)	(+3)	(+1)

( ): in brackets, percentage difference vs. controls.

No statistically significant differences vs. controls

 

Mean Body Weight Changes (g)

Dose level (mg/kg bw/day)	0	100	300	1000
Body weight change (g)
GD 2-5 (pre-treatment	+41	+29	+79	+38
GD 5-6 (pre-treatment)	+18	+4	+25	+25
GD 6-9	+27	+43	+39	-9
GD 9-12	+47	+52	+53	+33
GD 12-15	+72	+82	+75	+85
GD 15-18	+27	+10	+40	+25
GD 18-21	+22	+43	+27	+44
GD 21-24	+41	+36	+47	+46
GD 24-27	+54	+55	+61	+56
GD 27-29	+74	+66	+62	+80
GD 6-29	+364	+387	+405	+362

No statistically significant differences vs. controls.

 

Mean Food Consumption (g/day/animal)

Dose level (mg/kg bw/day)	0	100	300	1000
GD 2-3 (pre-treatment)	120	122	126	147
		(+2)	(+5)	(+23)
GD 3-4 (pre-treatment)	152	155	184	164
		(+2)	(+21)	(+8)
GD 4-5 (pre-treatment)	181	161	184	170
		(-11)	(+2)	(-6)
GD 5-6 (pre-treatment)	177	168	193	176
		(-5)	(+9)	(-1)
GD 6-7	161	167	173	135
		(+4)	(+7)	(-16)
GD 7-8	173	160	183	120**
		(-8)	(+6)	(-31)
GD 8-9	163	163	166	112#
		(0)	(+2)	(-31)
GD 9-10	164	165	171	115#
		(+1)	(+4)	(-30)
GD 10-11	160	172	173	128*
		(+8)	(+8)	(-20)
GD 11-12	151	161	164	125
		(+7)	(+9)	(-17)
GD 12-13	138	143	146	111
		(+4)	(+6)	(-20)
GD 13-14	137	142	143	115
		(+4)	(+4)	(-16)
GD 14-15	142	144	150	120
		(+1)	(+6)	(-15)
GD 15-16	148	152	160	134
		(+3)	(+8)	(-9)
GD 16-17	166	167	173	144
		(+1)	(+4)	(-13)
GD 17-18	171	169	168	150
		(-1)	--2)	(-12)
GD 18-19	157	156	151	138
		(-1)	(-4)	(-12)
GD 19-20	151	162	157	137
		(+7)	(+4)	(-9)
GD 20-21	151	153	153	140
		(+1)	(+1)	(-7)
GD 21-22	141	137	138	131
		(-3)	(-2)	(-7)
GD 22-23	130	129	135	136
		(-1)	(+4)	(+5)
GD 23-24	113	120	130	122
		(+6)	(+15)	(+8)
GD 24-25	111	114	119	123
		(+3)	(+7)	(+11)
GD 25-26	113	126	127	123
		(+12)	(+12)	(+9)
GD 26-27	127	129	120	121
		(+2)	(-6)	(-5)
GD 27-28	132	135	134	128
		(+2)	(+2)	(-3)
GD 28-29	140	135	138	143
		(-4)	(-1)	(+2)

( ): in brackets, percentage difference vs. controls;

Statistically significant differences vs. controls: *: p<0.05; **: p<0.01; #: p<0.001.

 

Summary of Gross Pathology Findings

	Females
Dose level (mg/kg bw/day)	0	100	300	1000
Number of Animals per Group	22	22	22	22
Thymus: reddish color	1
Stomach: colored areas		1
Gall bladder: abnormal color				1
Female gonads: serous cyst on connective tissue, periovarian region	2	1	5	3

No statistically significant differences vs. controls.

 

Mean Carcass Weights, Net Body Weight Changes and Gravid Uterus Weights (g)

Dose level (mg/kg bw/day)	0	100	300	1000	HCD [Min.; Max.]
Gravid uterus weight	575.5	523.5	582.5	576.1	[483.0; 589.5]
		(-9)	(+1)	(0)
Carcass weight	3292.9	3331.0	3384.4	3315.8	[3287.0; 3525.0]
		(+1)	(+3)	(+1)
Net body weight change from GD 6	-211.5	-136.6	-178.0	-214.4	[-296.9; -121.7]
		(-35)	(-16)	(+1)

( ): in brackets, percentage difference vs. controls.

No statistically significant differences vs. controls.

HCD: Historical Control Data, n = 8 studies (2019 to 2021).

 

Hysterectomy Data

Dose level (mg/kg bw/day)	0	100	300	1000	HCD [Min.; Max.]
Number of pregnant females at hysterectomy	19	21	21	21	183
Number of females with live fetuses at termination	19	21	21	21	165
Number of females with total resorption	0	0	0	0	0
Mean number of corpora lutea	10.9	10.4	12.0	11.1	[10.8; 13.0]
Mean number of implantation sites	9.8	8.9	10.2	9.9	[9.2; 10.6]
Mean pre-implantation loss (%)	9.5	14.4	13.8	10.8	[9.4; 21.7]
Mean number of live fetuses	9.4	8.6	9.7	9.4	[8.7; 9.9]
Dead fetuses (%)	0.0	0.0	0.4	0.0	[0.00; 0.39]
Mean number of implantation scars	0.0	0.0	0.0	0.0	/
Mean number of early resorptions	0.2	0.2	0.1	0.2	/
Mean number of late resorptions	0.3	0.0	0.3	0.3	/
Mean post-implantation loss (%)	4.4	3.5	4.4	4.8	[2.6; 13.2]

No statistically significant differences vs. controls.

HCD: Historical Control Data, n = 8 studies (2019 to 2021).

/: not available in HCD

 

 

FETAL TOXICITY

 

Mean Fetal Body Weights

Dose level (mg/kg bw/day)	0	100	300	1000	HCD [Min.; Max.]
Mean fetal body weight (male fetuses, g)	42.4	42.9	41.6	41.5	[38.9; 43.8]
		(+1)	(-2)	(-2)
Mean fetal body weight (female fetuses, g)	39.5	40.6	40.0	39.9	[37.0; 42.5]
		(+3)	(+1)	(+1)
Mean fetal body weight (male and female fetuses combined, g)	41.1	41.7	40.7	40.7	[37.8; 43.3]
		(+1)	(-1)	(-1)

( ): in brackets, percentage difference vs. controls.

No statistically significant differences vs. controls.

HCD: Historical Control Data, n = 8 studies (2019 to 2021).

 

Mean Percentages of Male Fetuses

Dose level (mg/kg bw/day)	0	100	300	1000	HCD [Min.; Max.]
Mean percentage of male fetuses (%)	50.6	53.7	47.6	53.0	[42.3; 55.3]
		(+6)	(-6)	(+5)

( ): in brackets, percentage difference vs. controls.

No statistically significant differences vs. controls

HCD: Historical Control Data, n = 8 studies (2019 to 2021).

 

Litter (L) and Fetal (F) incidences (%) of External Variations

Dose level (mg/kg bw/day)	0	100	300	1000	HCD [Min.; Max.]
Litters evaluated, n	19	21	21	21	163
Fetuses evaluated, n	178	181	205	198	1518
General
. local edema, L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.8 (0.5)	[0.0; 0.0]
Mouth, jaw, palate
. protruding tongue, L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.8 (1.0)	[0.0; 0.0]
Trunk
. enlarged abdomen, L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.8 (0.5)	[0.0; 4.8]

n: number.

No statistically significant differences vs. controls.

HCD: Historical Control Data, n = 8 studies (2019 to 2021).

 

Litter (F) and Fetal (F) incidences (%) of External Malformations

Dose level (mg/kg bw/day)	0	100	300	1000	HCD [Min.; Max.]
Litters evaluated, n	19	21	21	21	163
Fetuses evaluated, n	178	181	205	198	1518
Eyes
. open eye, L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.8 (1.0)	[0.0; 4.8]
Trunk
. omphalocele, L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.8 (0.5)	[0.0; 5.6]
Litters affected, n (%)	0 (0.0)	0 (0.0)	0 (0.0)	2 (9.5)	7 (4.3)
Fetuses affected, n (%)	0 (0.0)	0 (0.0)	0 (0.0)	3 (1.5)	8 (0.5)

n: number.

No statistically significant differences vs. controls.

HCD: Historical Control Data (litter incidences), n = 8 studies (2019 to 2021)

 

Litter (F) and Fetal (F) incidences (%) of External Malformations

Dose level (mg/kg bw/day)	0	100	300	1000	HCD [Min.; Max.]
Litters evaluated, n	19	21	21	21	163
Fetuses evaluated, n	178	181	205	198	1518
Brain
. cranial cavity: liquid content, L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.8 (1.0)	[0.0; 0.0]

n: number.

No statistically significant differences vs. controls.

HCD: Historical Control Data (litter incidences), n = 8 studies (2019 to 2021)

 

Litter (L) and Fetal (F) Incidences (%) of Soft Tissue Malformations

Dose level (mg/kg bw/day)	0	100	300	1000	HCD [Max.]
Litters evaluated, n	19	21	21	21	163
Fetuses evaluated, n	178	181	204	198	1518
Eye
. microphthalmia, L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	9.5 (2.5)	[5.0]
Brain
. marked dilated cerebral ventricle, L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.8 (1.5)	[4.5]
Heart
. cor triloculare, L (F)	5.3 (0.6)	0.0 (0.0)	0.0 (0.0)	4.8 (0.5)	[4.8]
Kidneys
. malpositioned kidney, L (F)	10.5 (1.1)	0.0 (0.0)	4.8 (0.5)	4.8 (0.5)	[0.0]
Vessels
. dilated aortic arch, L (F)	5.3 (0.6)	0.0 (0.0)	0.0 (0.0)	9.5 (1.0)	[5.6]
Ureter
. short ureter, L (F)	10.5 (1.1)	0.0 (0.0)	4.8 (0.5)	4.8 (0.5)	[0.0]
Litters affected, n (%)	3 (15.8)	0 (0.0)	1 (4.8)	3 (14.3)	18 (11.0)
Fetuses affected, n (%)	3 (1.7)	0 (0.0)	1 (0.5)	7 (3.5)	19 (1.3)

n: number.

No statistically significant differences vs. controls

HCD: Historical Control Data (litter incidences), n = 8 studies (2019 to 2021)

 

Litter (L) and Fetal (F) incidences (%) of Skeletal Cartilages

Dose level (mg/kg bw/day)	0	100	300	1000	HCD [Max.]
Litters evaluated, n	19	21	21	21	163
Fetuses evaluated, n	178	181	204	198	1518
Head-others
. cartilage of hyoid centrum: present, L (F)	0.0 (0.0)	4.8 (0.6)	4.8 (0.5)	9.5 (2.0)	[33.3]
Thoracic vertebra(e)
. cartilage of thoracic vertebra(e): fused, L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.8 (1.0)	[0.0]
. cartilage of thoracic vertebra(e): absent, L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.8 (1.0)	[4.3]
Caudal vertebra(e)
. cartilage of caudal vertebra(e): absent, L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.8 (0.5)	[9.1]
Rib
. cartilage of rib(s): absent, L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.8 (0.5)	[4.3]

n: number.

No statistically significant differences vs. controls

HCD: Historical Control Data (litter incidences), n = 8 studies (2019 to 2021)

 

Litter (L) and Fetal (F) incidences (%) of Skeletal Variations

Dose level (mg/kg bw/day)	0	100	300	1000	HCD [Max.]
Litters evaluated, n	19	21	21	21	163
Fetuses evaluated, n	178	181	204	198	1518
Head-others
. hyoid: incomplete ossification of centrum	0.0 (0.0)	4.8 (0.6)	4.8 (0.5)	9.5 (2.0)	[44.4]
Thoracic vertebra(e)
. unossified arch, L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.8 (0.5)	[0.0]
Sternebra
. unossified 5^th sternebra, L (F)	52.6 (13.5)	47.6 (15.5)	61.9 (15.2)	81.0 (26.8**)	[80.0]
. unossified 6^th sternebra, L (F)	15.8 (3.4)	9.5 (2.2)	14.3 (1.5)	28.6 (5.1)	[38.1]
. incomplete ossification of 1^st to 4^th sternebra(e) , L (F)	5.3 (0.6)	4.8 (0.6)	0.0 (0.0)	19.0 (2.0)	[28.6]
. extra sternebral ossification site, L (F)	5.3 (1.1)	4.8 (0.6)	0.0 (0.0)	9.5 (1.0)	[10.0]
Rib
. unossified rib(s) , L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.8 (0.5)	[0.0]

Statistically significant difference vs. controls **: p<0.01.

HCD: Historical Control Data (litter incidences), n = 8 studies (2019 to 2021).

 

Litter (L) and Fetal (F) Incidences (%) of Skeletal Malformations

Dose level (mg/kg bw/day)	0	100	300	1000	HCD [Min.; Max.]
Litters evaluated, n	19	21	21	21	163
Fetuses evaluated, n	178	181	204	198	1518
Head-skull
. nasal: split, L (F)	5.3 (0.6)	0.0 (0.0)	4.8 (0.5)	0.0 (0.0)	[0.0; 0.0]
Thoracic vertebra(e)
. fused, L (F)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.8 (1.0)	[0.0; 4.3]
. absent hemivertebra(e)	0.0 (0.0)	0.0 (0.0)	4.8 (0.5)	4.8 (0.5)	[0.0; 4.3]
Lumbar vertebra(e)
. absent	5.3 (0.6)	4.8 (0.6)	4.8 (0.5)	0.0 (0.0)	[0.0; 9.1]
. supernumerary	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	4.8 (0.5)	[0.0; 0.0]
Rib
. branched rib(s)	0.0 (0.0)	4.8 (0.6)	0.0 (0.0)	0.0 (0.0)	[0.0; 0.0]

n: number.

No statistically significant differences vs. controls

HCD: Historical Control Data (litter incidences), n = 8 studies (2019 to 2021)

Conclusions:

Under the test conditions of this study, the NOAEL (maternal and fetal) = 300 mg/kg bw/day.

Executive summary:

In a prenatal developmental toxicity study performed in accordance with OECD guideline 414 and in compliance with GLP, the test material diluted in PBS was administered through gavage to groups of New Zealand White time-mated rabbits (22/dose) at dose levels of 0 (vehicle control), 300 and 1000 mg/kg bw/day from Day 6 to 28 of pregnancy. The females were checked daily for mortality and clinical signs. Body weights were recorded at designated intervals. Food consumption was recorded daily. On GD 29, the females were euthanized and a macroscopic post-mortem examination was performed. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, uterine scars, early/late resorptions and live/dead fetuses were recorded. Placentas were observed. The fetuses were weighed and sexed by internal examination of the gonads. Detailed external, soft tissue and skeletal examinations (including cartilage) were performed in the fetuses. Macroscopic lesions were collected from the dams and preserved in 10% buffered formalin.

 

Maternal toxicity:

There were no unscheduled deaths and no test-item related clinical signs. There were no adverse effects on mean body weight at any dose-levels [only a minimal body weight loss at 1000 mg/kg bw/day (-9 g vs. +27 g in controls on GD 6-9) with a return towards control values from
GD 9-12]. Mean food consumption was low at 1000 mg/kg/day from the beginning of the treatment period (down to -31% vs. controls on GD 7-8 and GD 8-9 with p < 0.01 and 0.001, respectively) with a return towards control values from GD 22-23. Taking into account the amplitude of the differences and the duration of the effect, this finding was considered to be test-item related and adverse. At 300 and 100 mg/kg/day, there were no effects on mean food consumption. At necropsy, there were no test-item related macroscopic findings and no effects on mean gravid uterus weight, mean carcass weight, mean net body weight change or hysterectomy data (mean pre-/post-implantation losses and mean number of live fetuses).

 

Embryo-Fetal Toxicity:

There were no effects on mean fetal body weight or sex ratio (percentage of male fetuses). At 1000 mg/kg bw/day, 6 litters had fetuses with a series of malformations (external, soft-tissue and/or skeletal examinations) that were only observed at this dose level or with higher incidences when compared to controls or Historical Control Data. At 300 and 100 mg/kg bw/day, there were no test-item related malformations. From 100 mg/kg bw/day and when compared to controls, there were dose-related delays in ossification (i.e. variations), which were considered to represent non-adverse test item-related effects.

 

On the basis of the experimental conditions and results obtained in this study:

• the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 300 mg/kg bw/day based on the decreased mean food consumption (down to -31% controls ) at 1000 mg/kg bw/day,


• the NOAEL for embryo-fetal development was considered to be 300 mg/kg bw/day based on the increased incidence of fetal malformations at 1000 mg/kg bw/day.

 

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 June 2017 to 29 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch no. 20170320 provided by Solvay
- Expiration date of the lot/batch: 20 March 2018
- Purity test date: 20 March 2017
- Purity: 99.66%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 oC, below 70 RH%)
- Solubility and stability of the test substance in the solvent/vehicle: Test item formulation samples in the 10-100 mg/mL concentration range (using PBS as vehicle) were proven to be stable for at least 5 days when stored at room temperature (20±5oC).

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633 Sulzfeld, Germany) from SPF colony.
- Age at study initiation: Young adult female rats, nulliparous and non-pregnant, at least 15 weeks old at mating.
- Weight at study initiation: 205-278 g (the variation did not exceed ± 20% of the mean weight)
- Fasting period before study: no
- Housing: Standard laboratory conditions; individual housing
- Diet (e.g. ad libitum): The animals were provided with ssniff® SM Autoclavable Complete Diet for Rats/Mice – Breeding and Maintenance (Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany) ad libitum.
- Water (e.g. ad libitum): The animals were provided with tap water (in water bottles) ad libitum.
- Acclimation period: at least 33 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9-23.9°C (target: 22 ± 3°C)
- Humidity (%): 27-65% (target: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 19 June 2017 To: 18 July 2017

Route of administration:

oral: gavage

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on a trial formulation, Phosphate Buffered Saline (PBS) was selected as a suitable vehicle.
- Concentrations in vehicle: 10, 30 and 100 mg/L
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Lot/batch no.: 1864184 / 1868691 / 1868848

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test item formulations for concentration and/or homogeneity was performed using a validated GC-FID method. Top, middle and bottom samples were taken from the test item formulations two times during the study (during the first week of the treatment and once on the second half of treatment). Samples were taken in duplicate (0.1 mL/each), one set to analyse and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: approximately 2-3 hours
- Further matings after two unsuccessful attempts: until at least 24 sperm positive females/group were attained
- Verification of same strain and source of both sexes: not specified
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

From Gestation Day 6 to Day 19

Frequency of treatment:

Daily

Duration of test:

Caesarean section and necropsy performed on Gestation Day 20

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 mated females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were set based on the available information of the chemical nature and characteristics of the test item and available results of a reproduction/developmental toxicity screening test (see section 7.8.1)

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cage-side (general) clinical observations were made twice daily (at the beginning and end of each working day).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals at the onset of treatment (GD 6) then weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded with precision of ±1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes - Food was measured with precision of ± 1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption was also calculated for each interval, including GD 0-6, GD 6-20 and GD 0-20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes -
Examinations included:
- Gravid uterus weight: Yes - The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses.
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: The placentas were examined macroscopically.

Fetal examinations:

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

Other: Each foetus was weighed individually (accuracy ±0.01 g) and subjected to external examination, plus an additional examination of the great arteries. The gender of foetuses was determined according to the appearance of the anogenital distance

Statistics:

The statistical evaluation of data was performed with the program package SAS v9.2 in case of Provantis v.9, or SPSS PC+4.0 (SPSS Hungary Kft, Budapest) in the case of data tabulated in Excel, by an appropriate statistical method.

In case of the SAS v9.2 software package (within the validated Provantis system) the following decision tree was applied automatically for statistical evaluation of numeric data. The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests were adequate
.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then a non-parametric analysis was used. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

In case of the SPSS PC+4.0 program package, the heterogeneity of variance between groups was checked by Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, then Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test.

Indices:

MATERNAL DATA:
- Number of animals at test start, no. of animals surviving, no. of pregnant animals, no. of animals with total intrauterine mortality
- Clinical signs (by gestation day)
- Mortality (by gestation day)
- Body weight and body weight gain: mean ± S.D.
- Corrected body weight on GD 20 (body weight minus gravid uterine weight) and corrected body weight gain GD 0-20 (body weight gain (GD 0-20) minus gravid uterine weight): mean ± S.D.
- Net body weight change (body weight gain during the treatment period (GD 6-20) minus gravid uterine weight): mean ± S.D.
- Gravid uterine weight: mean ± S.D.
- Food consumption: mean ± S.D.
- Gross pathology findings, placenta findings

CAESAREAN SECTION AND NECROPSY DATA:
- Number of corpora lutea: mean ± S.D.
- Number of implantations: mean ± S.D.
- Number and percentage of live foetuses: mean ± S.D.
- Number and percentage of intrauterine mortality: mean ± S.D.
Classified according to time of death: preimplantation loss, postimplantation loss, early and late embryonic loss, as well as foetal death
- Preimplantation loss: %, group mean
- Postimplantation loss: %, group mean

FOETAL DATA:
- Sex distribution: %, group mean
- Foetal body weight (accuracy 0.01 g): mean * S.D.
- External abnormalities/litter: %, group mean
- Visceral abnormalities/litter: %, group mean
- Skeletal abnormalities/litter: %, group mean

Historical control data:

Yes. Data included as an Appendix in the study report (not detailed here)

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related effects or systemic clinical signs were noted in the treated animals of the Low (100 mg/kg bw/day), Mid (300 mg/kg bw/day) or High (1000 mg/kg bw/day) dose groups.

Mortality:

no mortality observed

Description (incidence):

No mortality was recorded for any animals in the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item related effect on body weight was observed in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day) when compared to control.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No statistically significant changes were observed in the mean daily food consumption of dams in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day, respectively) compared to the control value.
The food consumption of the Low, Mid and High dose groups was comparable with the Control group, the difference in the daily mean food consumption (calculated for the entire period of the study) was not larger than 8.4% in any dose groups when compared to the control.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment related observations were recorded for any evaluated animals in the study.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

There was no statistically significant difference in foetal death in any test item treated groups compared to the control.
The mean number of corpora lutea was comparable with the control in all test item treated groups. (See Summary of the intrauterine evaluation in Table 3).

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was no statistically significant difference in the postimplantation loss or total intrauterine mortality between the test item treated and control groups (See Summary of the intrauterine evaluation in Table 3).

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

(See Summary of the intrauterine evaluation in Table 3).

Early or late resorptions:

no effects observed

Description (incidence and severity):

The early and the late embryonic loss values of the test item treated groups were comparable with control (See Summary of the intrauterine evaluation in Table 3).

Dead fetuses:

no effects observed

Description (incidence and severity):

The number of death foetuses were 2, 0, 2, and 1 in 1, 0, 2 and 1 litters for the Control, Low, Mid and High dose groups, respectively (See Summary of the intrauterine evaluation in Table 3).

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

One hundred five females (26 for the Control, Low, and Mid dose groups, 27 for the High dose group, respectively) were mated in the study. The number of confirmed pregnant, evaluated dams was 19 in the Control group, 21 in the Low dose group (100 mg/kg bw/day), 18 in the Mid dose group (300 mg/kg bw/day) and 20 in the High dose group (1000 mg/kg bw/day). Summary of pregnancy data is seen in Table 2.

Other effects:

no effects observed

Details on maternal toxic effects:

No adverse effects reported up to the highest dose tested

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effects reported up to the highest dose tested

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean foetal weight per litter in the test item did not differ significantly from the control mean value (See Table 4: Examination of viable foetuses).

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The mean number of viable foetuses was comparable with the control mean in all test item treated groups (See Table 4: Examination of viable foetuses).

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups (See Table 4: Examination of viable foetuses).

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The total number of retarded foetuses (runts) as well as the number of affected litters was similar in all test item treated group as in the Control group, indicating no effect on this parameter (See Table 4: Examination of viable foetuses).

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

See Table 4: Examination of viable foetuses

External malformations:

no effects observed

Description (incidence and severity):

Based on the external findings the number of malformed / variant / intact foetuses were comparable with the control in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day, respectively). All of the external findings (variations) were consistent in general nature and incidence with the concurrent study control data / existing historical control data or showed incidental occurrence, therefore considered as incidental findings. The external abnormalities are summarized in Table 5.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Based on the skeletal findings the number of malformed / variant / intact foetuses were comparable with the control in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day, respectively). All of the skeletal findings correspond with the current historical control or the concurrent study control data, or were considered to be incidental findings without dose response. The observed skeletal abnormalities are summarized in Table 6.

Visceral malformations:

no effects observed

Description (incidence and severity):

Based on the visceral findings the number of malformed / variant / intact foetuses were comparable with the control in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day, respectively). All of the visceral findings (variations) were consistent in general nature and incidence with the concurrent study control data / existing historical control data or showed incidental occurrence, therefore considered as incidental findings. The observed visceral abnormalities are summarized in Table 7.

Other effects:

no effects observed

Description (incidence and severity):

Evaluation of placentas: No abnormalities were observed on the placentas of any animals in the Control, Low (100 mg/kg bw/day), Mid (300 mg/kg bw/day) or High (1000 mg/kg bw/day) dose groups.

Details on embryotoxic / teratogenic effects:

No adverse effecs reported up to the highest dose tested.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effecs reported up to the highest dose tested.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

 Dose formulation analysis:

 Samples for test item concentration and homogeneity determination of the dosing formulations were collected two times during the treatment period. Formulation stability was established before this study. The test item concentration was analysed using a GC-FID method.

 The mean concentration of all formulations were found to be in the range of 92-102% of their nominal concentrations (10, 30 and 100 mg/mL) and were found to be homogenous.

 No test item was detected in the vehicle control formulations (see Table 1).

Table 1: Summary of the dose formulation analysis

Formulation	20 June 2017		10 July 2017
	Mean measured concentration (mg/mL)	Relative to the nominal concentration (%)	Measured concentration (mg/mL)	Relative to the nominal concentration (%)
Control	not detectable	-	not detectable	-
10 mg/mL	9.16 ± 0.19	92	9.39±0.16	94
30 mg/mL	28.5 ± 0.81	95	29.9±0.73	100
100 mg/mL	92.2 ± 4.80	92	102±2.87	102

Table 2: Summary of pregnancy data

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of mated females	26	26	26	27
Pre-terminal death or euthanasia	0	0	0	0
Number of non-pregnant females	7	5	8	7
Number of females with ≤ 5 implantation sites	2	1	2	2
Number of evaluated females on GD20 (Caesarean section)	19	21	18	20

 

Table 3: Summary of the intrauterine evaluation

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of evaluated dams	19	21	18	20
Mean number of corpora lutea	11.8	11.0	10.1	11.2	NS
Preimplantation loss, mean	1.6	1.7	1.3	1.4	NS
Preimplantation loss (%), mean	13.03	15.74	12.90	12.76	NS
Mean number of implantations	10.2	9.4	8.8	9.8	NS
Early embryonic loss, mean	0.9	0.7	0.6	0.8	NS
Early embryonic loss (%), mean	12.24	12.43	8.58	10.27	NS
Late embryonic loss, mean	0.3	0.1	0.2	0.6	NS
Late embryonic loss (%), mean	2.36	1.59	2.49	8.04	NS
Dead foetuses, mean	0.1	0.0	0.1	0.1	NS
Dead foetuses (%), mean	0.88	0.00	1.17	0.63	NS
Postimplantation loss, mean	1.3	0.8	0.9	1.4	NS
Postimplantation loss (%), mean	15.48	14.02	12.24	18.93	NS
Total intrauterine mortality, mean	2.9	2.5	2.2	2.8	NS
Total intrauterine mortality (%), mean	27.00	25.50	22.60	27.97	NS
Viable foetuses, mean	8.9	8.6	7.9	8.4	NS

Notes: Most important parameters are shown in bold.

NS: Statistically not significant when compared to the vehicle control.

Table 4: Examination of viable foetuses

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of examined litters	19	21	18	20
Viable foetuses, mean	8.9	8.6	7.9	8.4	NS
Male foetuses, mean	4.3	4.2	3.2	4.0	NS
Female foetuses, mean	4.6	4.3	4.7	4.5	NS
Total number of foetuses	169	180	142	168	NS
Total number of male foetuses	82	89	57	79	NS
Total number of female foetuses	87	91	85	89	NS
Sex distribution (% of males / females)	49 / 51	49 / 51	40 / 60	47 / 53	NS
Mean foetal weight / litter (g)	2.747	2.874	2.818	2.806	NS
Number of foetuses with retarded body weight	4	10	2	2	NS
Number of affected litters (with runts)	3	4	2	2	NS

Notes: Most important parameters are shown in bold.

NS: Statistically not significant when compared to the vehicle control.

Table 5: Summary table of the external abnormalities

Parameter			Dose (mg/kg bw/day)							HC data
			Control	100		300		1000
Total number of examined litters			19	21		18		20		670
Total number of examined foetuses			169	180		142		168		6889
Total number of intact (normal) foetuses			168	179		142		168		--
Total number of foetuses / litters with malformation			0 / 0	1 / 1		0 / 0		0 / 0		--
Total number of foetuses / litters with variation			1 / 1	0 / 0		0 / 0		0 / 0		--
External malformations
Craniochisis and Holorachischisis	Litter incidence	n	0		1		0		0		1
		%	0.0		4.8		0.0		0.0		0.149
	Foetal incidence	n	0		1		0		0		1
		%	0.000		0.556		0.000		0.000		0.015
External variations
Subcutaneous oedema, localized	Litter incidence	n	1		0		0		0		3
		%	5.3		0.0		0.0		0.0		0.62
	Foetal incidence	n	1		0		0		0		3
		%	0.592		0.000		0.000		0.000		0.06

 Notes: Numbers represent the number of abnormalities / number of affected litters.

HC: historical control

No statistically significant differences were noted when compared to the control group.

Table 6: Summary table of the skeletal abnormalities

Parameter	Dose (mg/kg bw/day)				HC data
	Control	100	300	1000
Total number of examined litters	19	20	18	18	669
Total number of examined foetuses	85	90	70	84	3435
Total number of intact (normal) foetuses	78	76	63	77	--
Total number of foetuses / litters with malformation	0 / 0	1 / 1	1 / 1	0 / 0	--
Total number of foetuses / litters with variation	7 / 6	13 / 8	6 / 4	7 / 5	--

Notes:Numbers represent the number of abnormalities / number of affected litters.

HC: historical control

Table 7: Summary table of the visceral abnormalities

Parameter	Dose (mg/kg bw/day)				HC data
	Control	100	300	1000
Total number of examined litters	18	21	17	20	670
Total number of examined foetuses	84	90	72	84	3450
Total number of intact (normal) foetuses	79	81	70	78	--
Total number of foetuses / litters with malformation	1 / 1	4 / 4	1 / 1	1 / 1	--
Total number of foetuses / litters with variation	4 / 3	5 / 4*	1 / 1	5 / 3	--

Notes:Numbers represent the number of abnormalities / number of affected litters.

HC: historical control

No statistically significant differences were noted when compared to the control group.

*: One additional foetus with both malformation and variation was classified as foetus with malformation, and counted only in that category.

Conclusions:

In this prenatal developmental toxicity study in rats, from the observations made in the dams and their foetuses, no signs of maternal toxicity were noted and there was no toxicologically relevant adverse effect on embryos or foetuses in any test item treated groups. The no-observed-adverse-effect (NOAEL) levels maternal toxicity, embryotoxicity, foetotoxicity and teratogenicity was 1000 mg/kg bw/day.

Executive summary:

This developmental toxicity study was performed to assess the effects of the test itemAUGEO SL 191on the embryonic and foetal development (including the organogenesis period) of Hannover Wistar rats in their first pregnancy. The dams (one control and three test item treated groups) were treated daily by oral (gavage) administration, from gestation day 6 (GD 6) up to and including gestation day 19 (GD 19), where sperm positive day was counted as day 0 of pregnancy (GD 0). Control dams were treated with the vehicle (PBS) only. Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD 20.

 The doses were selected by the Sponsor based on available information of the chemical nature and characteristics of the test item and available results of a reproduction/developmental toxicity screening test [9]. In this study (OECD guideline No. 422), the NOAEL of the test item was 1000 mg/kg bw/day. Based on this information, the doses of 100, 300 and 1000 mg/kg bw/day were deemed suitable for the purpose of the study.

 Test item formulations were analysed for concentration and homogeneity two times during the treatment period using a validated GC-FID method. Simultaneously, vehicle control formulations were analysed for concentration.

 Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentas were examined macroscopically.

 The number of confirmed pregnant, evaluated dams was 19 in the Control, 21 in the Low (100 mg/kg bw/day), 18 in the Mid (300 mg/kg bw/day) and 20 in the High (1000 mg/kg bw/day) dose groups, respectively.

 

Results:

All test item formulations were within the range of 92-102%of nominal concentration and were found to be homogenous. No test item was detected in the vehicle control samples. Based on these results, test item formulations were considered suitable for the study purposes.

 There were no mortalities or clinical signs in the dams during the study, related to the test item treatment.

 There was no test item related adverse effect on maternal body weight / body weight gain, maternal corrected body weight / body weight gain, and on food intake in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day, respectively).

 There were no toxicologically significant differences, or test item related-changes in the examined parameters of the intrauterine development up to and including 1000 mg/kg bw/day.

 The mean number of viable foetuses in all test item treated groups as well as their sex distribution were comparable with the control mean.No test item related effect was observed on the mean foetal body weight per litter.

 No remarkable internal or external observations related to test item treatment were recorded for any pregnant animals duringnecropsy. No remarkable abnormalities were observed on the placentas in any examined groups.

 There were no test item related effects on external, visceral or skeletal development of foetuses in the study.

 

In conclusion, AUGEO SL 191,when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 induced no signs of maternal toxicity and no toxicologically relevant adverse effect on embryos or foetuses in any test item treated groups. The no-observed-adverse-effect (NOAEL) levels for maternal toxicity, embryotoxicity, foetotoxicity and teratogenicity was 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

GLP studies conducted according to OECD Guideline 414 without any deviation (Klimisch score = 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

Two prenatal developmental toxicity studies are available and considered as key studies:

1- In a prenatal developmental toxicity study performed in accordance with OECD guideline 414 and in compliance with GLP, the test material was administered through gavage to groups of Wistar rats at dose levels of 0 (vehicle control), 300 and 1000 mg/kg bw/day from Day 6 to 19 of pregnancy. Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentas were examined macroscopically. The number of confirmed pregnant, evaluated dams was 19 in the Control, 21 in the Low (100 mg/kg bw/day), 18 in the Mid (300 mg/kg bw/day) and 20 in the High (1000 mg/kg bw/day) dose groups, respectively.

 

 There were no mortalities or clinical signs in the dams during the study, related to the test item treatment.

 There was no test item related adverse effect on maternal body weight / body weight gain, maternal corrected body weight / body weight gain, and on food intake in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day, respectively).

 There were no toxicologically significant differences, or test item related-changes in the examined parameters of the intrauterine development up to and including 1000 mg/kg bw/day. The mean number of viable foetuses in all test item treated groups as well as their sex distribution were comparable with the control mean. No test item related effect was observed on the mean foetal body weight per litter. No remarkable internal or external observations related to test item treatment were recorded for any pregnant animals duringnecropsy. No remarkable abnormalities were observed on the placentas in any examined groups.

 There were no test item related effects on external, visceral or skeletal development of foetuses in the study.

 

Under the test conditions of this study, AUGEO SL 191 induced no signs of maternal toxicity and no toxicologically relevant adverse effect on embryos or foetuses in any test item treated groups. The no-observed-adverse-effect (NOAEL) levels for maternal toxicity, embryotoxicity, foetotoxicity and teratogenicity was 1000 mg/kg bw/day.

 

2- In a prenatal developmental toxicity study performed in accordance with OECD guideline 414 and in compliance with GLP, the test material diluted in PBS was administered through gavage to groups of New Zealand White time-mated rabbits (22/dose) at dose levels of 0 (vehicle control), 300 and 1000 mg/kg bw/day from Day 6 to 28 of pregnancy. The females were checked daily for mortality and clinical signs. Body weights were recorded at designated intervals. Food consumption was recorded daily. On GD 29, the females were euthanized and a macroscopic post-mortem examination was performed. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, uterine scars, early/late resorptions and live/dead fetuses were recorded. Placentas were observed. The fetuses were weighed and sexed by internal examination of the gonads. Detailed external, soft tissue and skeletal examinations (including cartilage) were performed in the fetuses. Macroscopic lesions were collected from the dams and preserved in 10% buffered formalin.

Maternal toxicity:

There were no unscheduled deaths and no test-item related clinical signs. There were no adverse effects on mean body weight at any dose-levels [only a minimal body weight loss at 1000 mg/kg bw/day (-9 g vs. +27 g in controls on GD 6-9) with a return towards control values from
GD 9-12]. Mean food consumption was low at 1000 mg/kg/day from the beginning of the treatment period (down to -31% vs. controls on GD 7-8 and GD 8-9 with p < 0.01 and 0.001, respectively) with a return towards control values from GD 22-23. Taking into account the amplitude of the differences and the duration of the effect, this finding was considered to be test-item related and adverse. At 300 and 100 mg/kg/day, there were no effects on mean food consumption. At necropsy, there were no test-item related macroscopic findings and no effects on mean gravid uterus weight, mean carcass weight, mean net body weight change or hysterectomy data (mean pre-/post-implantation losses and mean number of live fetuses).

Embryo-Fetal Toxicity:

There were no effects on mean fetal body weight or sex ratio (percentage of male fetuses). At 1000 mg/kg bw/day, 6 litters had fetuses with a series of malformations (external, soft-tissue and/or skeletal examinations) that were only observed at this dose level or with higher incidences when compared to controls or Historical Control Data. At 300 and 100 mg/kg bw/day, there were no test-item related malformations. From 100 mg/kg bw/day and when compared to controls, there were dose-related delays in ossification (i.e. variations), which were considered to represent non-adverse test item-related effects.

 

On the basis of the experimental conditions and results obtained in this study:

• the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 300 mg/kg bw/day based on the decreased mean food consumption (down to -31% controls ) at 1000 mg/kg bw/day,


• the NOAEL for embryo-fetal development was considered to be 300 mg/kg bw/day based on the increased incidence of fetal malformations at 1000 mg/kg bw/day.

 

Justification for classification or non-classification

No adverse signs of toxicity to reproduction (mating performance, fertility and post-natal development of offspring) were observed in rats in a combined repeated dose toxicity and reproduction / developmental toxicity screening test (OECD 422, GLP, K.1, 2013). 

Two prenatal developmental toxicity studies are carried out in rats and rabbits (OECD 414, GLP, K.1):

- In rat, no maternal toxicity was observed. No remarkable internal or external observations related to test item treatment were recorded for any pregnant animals during necropsy. No remarkable abnormalities were observed on the placentas in any examined groups. There were no test item related effects on external, visceral or skeletal development of foetuses in the study. The NOAEL (Developmental) is 1000 mg/kg bw/day.

- In rabbit, only decreased of mean food consumption (down by 31% as compared to controls) at 1000 mg/kg bw/day was observed. At necropsy, there were no test-item related macroscopic findings and no effects on mean gravid uterus weight, mean carcass weight, mean net body weight change or hysterectomy data (mean pre-/post-implantation losses and mean number of live fetuses). There were no effects on mean fetal body weight or sex ratio (percentage of male fetuses). At 1000 mg/kg bw/day, six litters had fetuses with a series of malformations (external, soft-tissue and/or skeletal examinations) that were only observed at this dose level or with higher incidences when compared to controls or Historical Control Data. At 300 and 100 mg/kg bw/day, there were no test-item related malformations. From 100 mg/kg bw/day and when compared to controls, there were dose-related delays in ossification (i.e. variations), which were considered to represent non-adverse test item-related effects. Based on the results of this study, the NOAEL for embryo-fetal development was considered to be 300 mg/kg bw/day based on the increased incidence of fetal malformations at 1000 mg/kg bw/day.

According to the CLP criteria, categories 1B and 2 are based on:

• animal data,


• absence of other toxicity effects or if there are other toxic effects, the reprotoxicity effects are not considered as secondary non specific effects

The difference between the 2 categories is based on:

• clear evidence of adverse effects on reproduction and human relevance for the category 1B


• Some evidence of adverse effects on reproduction but not sufficiently convincing and/or doubt about the human relevance for the category 2

The effects observed with Augeo SL191 are based on an animal study and in absence of obvious maternal toxicity.

Even though the effects (mainly microphthalmia) are considered to be clear evidence of adverse effects on the development, the category 2 was preferred because the human relevance is not warranted due to:

• The effects are possibly rabbit-specific since they are only observed in the rabbit and not in the rat,


• The effects are only observed at the highest dose (1000 mg/kg/day), which can sometimes overwhelm the normal metabolization capacity of the organisms,


• The hydrolysis study (OECD 111) performed on Augeo SL191 indicated that the substance degrades quickly at 35°C particularly at acid pH (pH=4). Some internal stability tests also indicated that Augeo SL191 degraded quickly (c.a in 1 hour) at pH=2, a pH value corresponding to rabbit stomach conditions. In this last test, Augeo SL 191 transformed quickly in acetone and glycerol which do not raise concerns of reprotoxicity so far.    

Based on this information observed in rabbit species at the highest dose tested, 2,2-Dimethyl-1,3-dioxolane-4-methanol is classified as reprotoxic category 2; H361d (Suspected of damaging the unborn child) according to Regulation (EC) N° 1272 -2008 (CLP) and UN-GHS.

It should be noted that this self-classification is transient and will be reassessed when more data become available. As category 2 has been identified, it is of importance to further investigate whether the substance is reprotoxic or not. That is why Solvay decided to launch an in vivo mechanistic reprotoxicity study on rabbits, to gather further information on the maternal toxicity (e.g. hepatotoxicity) and to investigate the toxicokinetic and metabolism behaviours of the substance. The latter is of importance due to the known lack of stability of Augeo SL191 in acidic conditions found in animal stomachs. In addition, more specific modes of action will be explored if possible such as the impact on the vitamin A pathway which is relevant for the type of effects seen in the study.

Results of this mechanistic study will be provided in the next update of this dossier (c.a November 2023).