2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3-yl]benzoxazole-5-sulphonamide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Screening: Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material is at least 750 mg/kg/day for parental and reproductive toxicity.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 October 2016 to 08 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI (Han)

Details on species / strain selection:

One of the rodent species acceptable to the regulatory authorities. Historical control data for the strain are available at the testing facility

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Males approximately 11 weeks and females approximately 13 weeks
- Weight at study initiation: males 344 to 411 g and females 209 to 276 g
- Fasting period before study: No
- Housing: Animals were housed in one air-conditioned room in a barrier protected unit. Males were housed in groups of 5 during the pre-mating and gestation periods. Females were housed in groups of 5 during the pre-mating period. Males and females were housed together during mating (one per sex per cage) and subsequently females were housed individually during gestation and with their litter during lactation.
Animals were housed in plastic cages, in compliance with European Regulations. Cellulose bedding or dust-free sawdust made from spruce tree wood was provided. A small amount (handful) of shredded paper was provided as enrichment for all animals. Any isolated animals had free access to a wooden gnaw block. Furthermore, tissues were also provided as enrichment for females towards the end of gestation.
- Diet: Rat pelleted complete diet ad libitum
- Water: Softened and filtered (0.2 μm) mains drinking water was available ad libitum (via an automatic watering system or bottles).
- Acclimation period: 8 days between animal arrival and start of treatment (males) or start of pre-test oestrous cycle smears (females).

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: > 35 %
- Air changes: At least 10 air changes per hour
- Photoperiod: 12 hours of light (artificial)/12 hours of darkness

IN-LIFE DATES
- From: Not specified
- To: 08 February 2017

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Water for injection

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was prepared as a suspension in the vehicle (water for injection). The test material was prepared at least once weekly and stored at 2 to 8 °C between uses. Formulations were maintained under continuous stirring for at least 15 minutes before and throughout the dosing procedure.

DOSE VOLUME: 10 mL/kg/day

VEHICLE
- Concentration in vehicle: 10, 30 and 75 mg/mL

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: After 2 weeks of treatment, animals were paired on the basis of one male and one female from the same group for a maximum of 14 days.
- Proof of pregnancy: The day of mating was confirmed by the presence of sperm in a vaginal smear and was recorded and taken as day 0 of gestation (G 0).
- After successful mating each pregnant female was caged (how): Mated females were separated from the males once mating had been confirmed and smearing ceased and housed individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

STABILITY
Suspensions at 1 and 200 mg/mL were demonstrated to be stable for 8 days when stored refrigerated (+2 to +8 °C), based on the results of a study at the testing facility.

ANALYSIS OF PREPARATIONS
Quadruple (4 x 1 g) accurate samples were taken from each formulation, including the vehicle, used on the first day of treatment of the main study phase. Samples were taken from the top, middle and bottom levels and analysed for accuracy. Groups 2 and 4 were also assessed for homogeneity. One set of samples (2 x 1 g) was analysed at the testing facility and the other set (2 x 1 g) was stored frozen at -25 to -15 °C.

- Method
UPLC (pump, autosampler, detector): Acquity HClass , Waters
Column: Waters, Sunfire C18, 50 mm x 4.6 mm x 2.5 µm, reference 186003417
Precolumn: Not applicable
Pre-injection volume: Not applicable
Oven temperature: 30 °C
Injection volume: 10 µL
Run time: 9 min
Autosampler temperature: 20 °C
UV Detection: 210 ± 4.8 nm
Retention time: around 4.1 minutes
Flow rate: 1 mL/minute
Mobile Phase A: 0.2 % formic acid in water
Mobile Phase B: 0.2 % formic acid in acetonitrile
Elution gradient: At 0 and 2 minutes, 60 % A, 40 % B; at 7 and 7.1 minutes, 40 % A, 60 % B; at 9 minutes, 40 % A, 60 % B
Wash: 50/50 CH3CN/H2O
Purge/SW: 50/50 CH3CN/H2O
Correction factor for test material: 100 %

- Calibration range and Quality Control (QC)
16, 25, 40, 60 and 80 µg/mL prepared from two independent standard solutions. Standard solution was prepared at 200 µg/mL in dilution solvent (50/50 Acetonitrile/DMSO). Dilution factors were 2/25, 1/8, 1/5, 3/10 and 2/5.
The formulation concentration range was validated at 1 to 200 mg/mL in water for injection.

VALIDATION
The method was validated by the testing facility and a pass was achieved for the following method conformities:
- Quadratic regression of the calibration curves (r ≥0.999);
- Standard solution conformity between calibration and QC stock solutions (± 2 %);
- Analysis conformity: of calibration solutions (SE) recovery within 95-105 % of the exact concentrations (except for LLOQ samples 90-110 %);
- QC solutions: recovery within 95-105 % of the exact concentrations (except for LLOQ QC samples 90-110 %). One QC sample may be outside the acceptance criteria.

FORMULAE
- Quadratic regression of the calibration curves:
y = Cx2 + Bx + A
Where:
y = test material peak area (µAUs)
x = concentrations of the test material calibration solution (µg/mL)

- Back calculated concentration (µg/mL):
[-B + √(B2 - 4C(A-y ))] / 2C

- Standard solution conformity: Deviation between the Calibration standard solutions (SS Cal) and Quality Control standard solutions (SS QC)
Deviation (%) = [(SS Cal area / SS QC area) - 1] x 100

- Analysis conformity, the recovery of the back-calculated concentration of SE or SQC:
Recovery (%) = (Individual back calculated concentration / nominal concentration) x 100

- Concentrations of the formulation samples (suspension):
Concentration (mg/mL) = (Back calculated conc x V x F x d) / (sample weight (g) x 1000)
Where:
V = volume of the volumetric flask used
d = formulation density
F = dilution factor

- Deviation from the nominal concentration of the formulations:
Deviation (%) = [(mean of experimental concentration - nominal concentration) / nominal concentration] x 100

RESULTS
The acceptance range for achieved concentrations was ± 15 % from the nominal concentration for formulations at 10, 30 and 75 mg/mL.
All formulations at 10, 30 and 75 mg/mL in vehicle (water for injection) including the vehicle, used on the first day of treatment of the main study phase, were in agreement with acceptance criteria. The formulations at 10 and 75 mg/mL tested for homogeneity were homogenous. The deviations from the nominal concentrations ranged from -9.8 % to -1.9 % and the RSD was ≤2.3 %. No significant amount of test material was detected in the vehicle sample.

Duration of treatment / exposure:

Males were dosed for 30 days, i.e. 14 days prior to mating, throughout the mating period and up to the day prior to necropsy. The first day of dosing is designated as Day 1.
Females that delivered were treated for at least 51 days, i.e. during 14 days prior to mating (the first day of dosing is designated as Day 1), the variable time to conception (the first day of gestation is designated as G 0), the duration of the pregnancy and 13 days after delivery (the first day of birth is designated as L 0), up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for at least 41 days.

Frequency of treatment:

Once daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

750 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Rationale for the dose selection was based on the results of the range-finder in which administration of the test material at doses of 500 and 1000 mg/kg/day was associated with a minimal effect on body weight gain in the high dose group with no obvious correlation effect on food consumption. For the main study phase, a high dose of 750 instead of 1000 mg/kg/day was employed to limit maternal toxicity since in the main phase, the animals were pregnant and assumed to be likely more sensitive than non-pregnant animals. Moreover, the duration of the treatment was defined to be at least 51 days (depending on the duration of the mating period).
- Animal assignment: Performed during the acclimatisation period, using a computer-generated randomisation. The mean body weights of each group at allocation were not statistically significantly different from each other (analysis of variance), each sex being considered separately.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All adults were observed twice daily, at the beginning and at the end of each working day (including weekends and public holidays) for morbidity/mortality. The animals were observed daily for clinical signs. To detect any clinical signs or reactions to treatment, the animals were observed before and once after dosing (based on information provided from the range-finder).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A full clinical examination was performed on each weighing day. Towards the end of the gestation, females were examined daily for signs of parturition.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed at the time of randomisation, on the first day of exposure (prior to the first exposure) and weekly thereafter during pre-mating and mating periods. Mated females were weighed on days 0, 4, 7, 11, 14, 17 and 20 of gestation and on days 1, 4, 7 and 13 of lactation.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption of males was recorded weekly during the pre-mating period. Food consumption of females was recorded for the following periods: weekly during the pre-mating period, days 0 to 4, 4 to 7, 7 to 11, 11 to 14, 14 to 17 and 17 to 20 of gestation and days 1 to 4, 4 to 7 and 7 to 13 of lactation.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the pre- mating period (Day 14) blood was withdrawn from the retro-orbital sinus. Samples were collected in K2-EDTA tubes for haematology parameters and in tubes containing trisodium citrate for coagulation parameters.

- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes, for about 17 hours
- How many animals: First 5 animals/sex/group
- Parameters examined for haematology: Haemoglobin, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, packed cell volume, red blood cell count, mean corpuscular volume, reticulocyte count, platelet count, total white blood cell count and differential white blood cell count.
-Parameters examined for coagulation: Prothrombin time, activated partial thromboplastin time and fibrinogen.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the pre- mating period (Day 14) blood was withdrawn from the retro-orbital sinus. Samples were collected in tubes without anticoagulant.
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes, for about 17 hours
- How many animals: First 5 animals/sex/group
- Parameters examined: Sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, total cholesterol, total bilirubin, total protein, albumin, creatinine, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase and bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Males were examined on Day 30 (last day of dosing), the day before scheduled necropsy. Females were examined on PND 12 (penultimate day of dosing) or PND 13 (last day of dosing), 2 or 1 day(s) before scheduled necropsy, respectively.
- Dose groups that were examined: 5 selected animals per sex per group (females selected had live pups); the same animals were selected for terminal investigations.
- Battery of functions tested: Auditory reflex, pupillary reflex, righting reflex, fore- and hind-limb grip strength and locomotor activity in an open field test.
In the locomotor activity in an open field test, activity was monitored by a video image analysis system (Videotrack supplied by Viewpoint, Lyon, France). The arena is divided into nine equal invisible regions; the time spent by the animal in each type of region (i.e. corner, centre or lateral) was recorded. Motor activity is divided into three categories: ambulatory activity (in which the centre of the image moves at more than 7 cm/sec), small movements (including grooming etc.) and inactivity. The proportion of time spent engaged in each type of activity and the total distance travelled by the rat were calculated.

IMMUNOLOGY: No

HORMONE ANALYSIS: Yes
- Time schedule for collection of blood: Terminal blood sampling. For males, this was after 30 days of treatment; for F0-females on lactation day 14; for unmated F0-females 28 days after the last day of the mating period; and F0-females (non-pregnant and the one with total litter death) on Day 26 of gestation. Blood samples of approximately 0.9 mL (target volume) were withdrawn from a retro-orbital sinus. Samples were collected in tubes without anticoagulant, between 7:00 and 9:00 a.m. because of diurnal variation of hormone concentrations. The samples were centrifuged at 1800 g, at approximately 4 °C for 10 minutes and the serum was separated in two aliquots (at least 150 μL for the first aliquot and the remainder for the second aliquot). Serum samples were stored deep-frozen (between -70 and -90 °C) and allowed to thaw prior to analysis.
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: No
- How many animals: All males and females (except group 3 female no. 152 which was found dead on Day 14) on the day of necropsy.
- Parameters examined: T4 was examined in all males; T4 was examined in the females if required. TSH levels were measured in all animals if required.

PREGNANCY AND PARTURITION: Yes
- Time schedule for examinations: The following was recorded for each female: date of mating, date of parturition (day 0 of lactation or L 0), duration of gestation and abnormalities of nesting or nursing behaviour.

Oestrous cyclicity (parental animals):

Daily vaginal smears were performed to determine the stage of oestrus beginning 14 days prior to treatment, the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal smears continued for any female with no evidence of copulation until termination of the mating period. On the day of scheduled necropsy, a vaginal smear was taken to determine the stage of the oestrous cycle and allow correlation with histopathology of female reproductive organs.

Sperm parameters (parental animals):

Parameters examined: For all males of each group, epididymides, prostate, seminal vesicles and testes were weighed. Paired organs were weighed together. During histopathological examination, the testes from the 5 randomly selected adult males of groups 1 (control) and 4 (high dose) and all males suspected to be infertile were examined. A detailed, qualitative evaluation of the testes was performed, taking into account the tubular stages of the spermatogenic cycle. The cell- or stage-specificity of any testicular findings was recorded, where appropriate.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
The size of each litter was adjusted to 8 pups on PND 4 by eliminating extra pups by random selection to yield 4 male and 4 female pups per litter where possible. Blood samples were collected from two surplus pups where possible, pooled and used for determination of serum T4 levels. No pups were eliminated when litter size dropped below the culling target (8 pups/litter). When there was only one pup available above the culling target, only one pup was eliminated and used for blood collection for possible serum T4 assessments.

PARAMETERS EXAMINED
-The following were recorded for each F1 litter: number of pups born (live and dead); external abnormalities of the pups; number, weight and sex of pups alive on PND’s 1, 4, 7 and 13; anogenital distance (normalised to the cube root of body weight) on PND 1; and presence of areola/nipples on PND 13 for all males in each litter.

GROSS EXAMINATION OF DEAD PUPS: Yes
Pups that died were necropsied. Any external abnormalities observed were recorded. Their stomach was examined for the presence of milk, if possible. Defects or cause of death was evaluated, if possible.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

HORMONE ANALYSIS: Yes
- Time schedule for collection of blood: Terminal blood sampling. On PND 4, blood samples of approximately 0.4 mL (target volume) were obtained by decapitation under anaesthesia. Blood samples from the pups per litter were collected into one serum tube. When the target volume of 0.4 mL could not be reach by pooling 2 pups, extra pups were sampled if available. When only 1 surplus pup per litter was available at culling, as much blood as possible was collected from this single pup. On PND 13, blood samples of approximately 0.9 mL (target volume) were withdrawn by intracardiac puncture under anaesthesia. Samples were collected in tubes without anticoagulant, between 8:00 and 10:30 a.m. because of diurnal variation of hormone concentrations. The samples were centrifuged at 1800 g, at approximately 4 °C for 10 minutes and the serum was separated into an aliquot of at least 150 μL on PND 4 and into two aliquots (at least 150 μL for the first aliquot and the remainder for the second aliquot) on PND 13.
Serum samples were stored deep-frozen (between -70 and -90 °C) and allowed to thaw prior to analysis.
- Anaesthetic used for blood collection: Yes, sodium pentobarbitone
- Animals fasted: No
- How many animals: For 2 pups per litter where possible on PND4 and two pups per litter where possible on PND 13 (one male and one female on each occasion where possible).
- Parameters examined: T4 was examined in all PND 13 pups and PND 4 pups if required. TSH levels were measured in PND13 pups if required.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
Surviving adult animals were killed by carbon dioxide inhalation followed by exsanguination. Group 1 female no. 111 with total litter death was killed by carbon dioxide inhalation and exsanguination then necropsied. Group 3 female no. 152 found dead on Day 14 was also necropsied. Any such females found after the first day of pairing were given a caesarean section to determine pregnancy status, number of corpora lutea and numbers and types of uterine implantations.
All animals were submitted to a macroscopic examination for structural or pathological changes, with special attention being paid to the reproductive organs. The uterus of all adult females was placed in ammonium sulphide solution in order to confirm the number of implantation sites.
- Males: Necropsy took place following completion of the mating period (after 30 days of dose administration).
- Females: animals which delivered were subjected to necropsy on day 14 of lactation, females which failed to deliver on post-coitum Day 26 (females with evidence of mating) or 28 days after the last day of the mating period (female without evidence of mating). For the female with total litter loss, necropsy took place within 24 hours of litter loss. In the case of spontaneous death, necropsy took place on the day of death.
Five animals per group were subjected to full macroscopic examination and recording of organ weights. The females selected all had live pups.

The following organ weights were recorded for the selected animals: Adrenal glands, brain, cervix and uterus, coagulating gland, epididymides, heart, kidneys, liver, ovaries, parathyroid and thyroid glands, prostate, seminal vesicles, spleen, testes and thymus.
For the other animals of each group, epididymides, ovaries, prostate, seminal vesicles, testes, thyroid and uterus were weighed. Paired organs were weighed together. The organs were weighed after dissection of fat and other contiguous tissues.

HISTOPATHOLOGY: Yes
All organs/tissues listed below were sampled for the 5 randomly selected adult males and females of each group (for females, only those with live pups) and for group 3 female no. 152 found dead on Day 14. Only a limited list of organs/tissues was sampled for all remaining animals (males and females, including females that failed to produce a viable litter by day 26 post coitum, or with total litter death).
All organs/tissues sampled were fixed and preserved in 10 % neutral formalin with the exception of Harderian glands, testes, epididymides, eyes and optic nerves which were fixed in modified Davidson's fluid. The same sampling and trimming procedures were used for all applicable tissues in all applicable dose groups. Blocks were prepared only for tissues which were evaluated histopathologically.
Histopathological examinations were performed as follows:
- For all organs/tissues from group 3 female no. 152 found dead on Day 14
- For all organs/tissues from the 5 randomly selected adult males and females of groups 1 (control) and 4 (high dose) (for females, only those with live pups)
- For the reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups
- For the testes from the 5 randomly selected adult males of groups 1 (control) and 4 (high dose) and all males suspected to be infertile.
Organs and tissues examined were: macroscopic lesions, adrenal glands, bone (femur) and articulation, bone (sternum) with bone marrow, brain, caecum, cervix, clitoral gland, coagulating gland, colon, duodenum, epididymides, eyes, Harderian glands, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (mandibular and mesenteric), mammary gland (inguinal region with skin), oesophagus, optic nerves, ovaries, parathyroid glands, Peyer’s patches, pituitary gland, preputial gland, prostate, rectum, sciatic nerve (left), seminal vesicles, skeletal muscle, skin, spinal cord (cervical thoracic and lumbar), spleen, stomach, testes, thymus, thyroid glands, trachea, urinary bladder, uterus and vagina.
All sections were stained with haematoxylin and eosin. Additional PAS staining was prepared for the testes from the 5 randomly selected adult males of groups 1 (control) and 4 (high dose) and all males suspected to be infertile. A detailed, qualitative evaluation of the testes was performed, taking into account the tubular stages of the spermatogenic cycle. The cell- or stage-specificity of any testicular findings was recorded, where appropriate.

Postmortem examinations (offspring):

GROSS PATHOLOGY: Yes
On PND 4 and PND 13, pups were killed by an intraperitoneal injection of sodium pentobarbitone. Pups culled were necropsied on PND 4. Pups surviving to terminal sacrifice were necropsied on PND 13.
Each pup was sexed and examined for external defects with special attention being paid to the external reproductive organs. The thyroid glands of one male and one female by litter were fixed and preserved in 10 % formalin. Any external abnormalities observed were recorded but not preserved.

HISTOPATHOLOGY: No

Statistics:

Analyses were performed by the Provantis acquisition system. The best transformation for the data (none, log or rank) was determined depending upon the kurtosis of the data and the probability of the Bartlett's test for homogeneity of the variances and an assessment of whether the size of the groups were approximately equal or not.
Non- or log-transformed data were analysed by parametric methods. Rank transformed data were analysed using non-parametric methods.
Data were then analysed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non-parametric data.
If no trend was found and means were not homogeneous, the data were analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
Selected incidence data were analysed using the Provantis data acquisition system and/or an SAS software package. A chi2 test was used for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.
The locomotor activity in an open field and pre-coital interval data were analysed using an SAS software package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk's test was used to assess the normality of the data distribution in each group. Data with homogenous variances and normal distribution in all groups were analysed using ANOVA followed by Dunnett’s test. Data showing non-homogenous variances or a non-normal distribution in at least one group were analysed using Kruskal-Wallis test followed by the Wilcoxon's rank sum test.
Microsoft Excel® (version 2003 or higher) was employed to present certain results and perform associated calculations.

Reproductive indices:

Pre-coital interval (in days): sum of days until successful insemination / number of inseminated females
Male and female copulation index (in %): (number of inseminated females / number of paired animals) x 100
Male and female fertility index (in %): (number of pregnant females / number of inseminated females) x 100
Pre-birth loss (in %): [(number of implantations - number of offspring born) / number of implantations] x 100

Offspring viability indices:

Live birth index (in %): (number of pups born alive / number of pups born) x 100
Viability index (in %): (number of pups alive on PND 4 / number of pups alive at birth) x 100
Lactation index (in %): (number of pups alive on PND 13 / number of pups alive on PND 4 (after culling)) x 100
Sex ratio (proportion of male pups in %): (number of males / number of pups) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related clinical signs in any group. Yellow-coloured faeces were noted in treated groups at 300 and 750 mg/kg/day. This was attributed to the yellow colour of the test material. Control female no. 113 had marked piloerection associated with decreased activity on lactation Day 1. These signs were most probably due to parturition and were no longer observed thereafter. Other incidental clinical observations included localised hairloss, chromodacryorrhea and/or scabs.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no test material-related deaths in any group. Control female no. 111 was euthanised on gestation Day 26 due to total litter resorption. Female no. 152 given 300 mg/kg/day was found dead on Day 14 during the pre-mating period following anaesthesia for blood sampling. In view of their nature and incidence, these isolated deaths were considered incidental.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes in body weights or body weight gains in males or females that were considered to be related to the test material during the pre-mating, gestation or lactation periods. Any differences noted in mean body weight gains were incidental since they were either not dose-related, transient or since mean values in treated groups were superior to those in the concurrent controls.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically-relevant changes in food consumption in males or females during the pre-mating, gestation or lactation periods. Mean food consumption was slightly lower in treated than in control females during the pre-mating period (up to -4.8 % in the 750 mg/kg/day group). Since this change was transient and not observed during the gestation or lactation periods and had no adverse effect on body weights, it was not considered toxicologically relevant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences noted in haematological and coagulation parameters between control and treated rats of both sexes that were considered to be toxicologically relevant.
Statistically significant dose-related slightly increased mean prothrombin time and dose-related slightly decreased mean fibrinogen concentration were noted on Day 14 for males only when compared with concurrent controls. Mean values remained, however, within the historical control ranges. These findings were therefore of no toxicological relevance.
Although differences in mean values attained statistical significance for females compared with the concurrent controls, the decrease in mean white blood cell and lymphocyte counts was of no toxicological significance in view of its low magnitude. In addition, mean values were within the historical control range.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences noted in serum clinical chemistry parameters between control and treated rats of both sexes that were considered to be toxicologically relevant.
Statistically significant dose-related slightly decreased mean sodium concentration was noted on Day 14 for both sexes. Dose-related slightly decreased mean cholesterol levels were noted on Day 14 for males only when compared with concurrent controls. All mean values remained, however, within the historical control ranges. These findings were therefore of no toxicological relevance.
The dose-related decrease in mean aspartate aminotransferase (ASAT) levels was due to a single male in the control group (no. 145) with a high ASAT value compared with other control males.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed.
Fore- and hind-limb grip strength mean values were lower than controls for males at 750 mg/kg/day and higher than controls for treated females. This was considered to be due to intra-group variability and therefore incidental.
The variation in motor activity (open field) did not indicate a relation with treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There was no evidence of any test material-related microscopic findings in animals given 750 mg/kg/day.
The nature or incidence of histological findings in the organs examined did not indicate any relationship to treatment and they were considered to be incidental and part of the normal background changes normally encountered in reproductive/developmental studies in rodents.

- Premature Decedents
Female no. 152 given 300 mg/kg/day was found dead on Day 14 during the pre-mating period following isoflurane anaesthesia for blood sampling. At the necropsy, a bilateral dilated kidney was noted, correlating histologically with slight pelvic dilatation, considered to be incidental and unrelated to treatment.
Control female no. 111 was euthanised due to total litter resorption. Histological evaluation of genital tract showed a normal oestrous cycle.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

T4 HORMONE ANALYSIS
No differences in Total T4 levels were noted among the different groups of F0-males.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the oestrous cycle were not affected by treatment with the test material.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

When compared with controls, there were no test material-related changes in reproductive organ weights associated with the administration of the test material. There was no evidence of any test material-related microscopic findings in reproductive organs in animals given 750 mg/kg/day.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test material-related influence on mating performance of the males and females in any group. All surviving animals mated with the exception of one pair in the 100 mg/kg/day group. Most mated females showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal oestrous cycle), with the exception of one female in each of the control (no. 118) and 300 mg/kg/day (no. 154) groups with a pre-coital interval of 9 and 14 days, respectively. The mean pre-coital interval was therefore slightly higher in these two groups but this was considered incidental.
There was no effect of treatment on fertility of either sex in any group. There was one female in each of the control and 100 mg/kg/day groups (nos. 118 and 136, respectively) that did not become pregnant. These isolated cases, including controls, were considered incidental.

- Litter data
Parturition: There were 8, 8, 9 and 10 females that successfully completed delivery in the control, 100, 300 and 750 mg/kg/day groups, respectively. The mean duration of gestation was comparable in all groups.
Number of implantation sites: The mean number of implantation sites was comparable in all groups.
Pre-birth loss: The percentage of pre-birth loss was not affected by treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed that were attributed to treatment with the test material

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no pup observations that suggested any association with maternal treatment. Incidental occasional findings included weak or thin pups, incomplete hair growth or haematoma.
The mean percentage of males per litter was abnormally low in the control group (30.8 %). This was considered incidental.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There were no test material-related differences in pup viability. The number of offspring born alive compared to the total number of offspring born was not affected by treatment. There was one pup dead or missing in the control group versus none in the treated groups.
The number of live offspring on Day 4 before culling compared to the number of offspring alive at birth was not affected by treatment. Two, three and two pups in the control, 300 and 750 mg/kg/day groups were found dead or missing versus none in the 100 mg/kg/day group between postnatal Days 0 and 4.
The number of live offspring on Day 13 compared to the number of offspring on Day 4 (post culling) was not affected by treatment. There was one pup found dead or missing in the 300 mg/kg/day group versus none in the other groups.
No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no obvious test material-related changes in pup body weights. The mean pup body weights (males and females) were slightly lower in the 300 and 750 mg/kg/day groups than in the control and 100 mg/kg/day groups from days 1 to 13 of lactation. These minor differences were considered to be due to the slightly higher live litter size at birth in the treated groups and not to treatment with test material.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no observations that were considered to be associated with administration of the test material.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

T4 HORMONE ANALYSIS
No differences in Total T4 levels were noted among the different groups of PND 13 pups.

LITTER SIZE
There were no test material-related differences in litter size. The mean litter size at birth was higher in the 300 and 750 mg/kg/day groups than in the control and 100 mg/kg/day groups. This was considered incidental.

MALE PUPS AREOLA/NIPPLES
There were no test material-related effects on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

ANOGENITAL DISTANCE
Anogenital distance (normalised for body weight) in male and female pups was not affected by treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed that were attributed to treatment with the test material

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material is at least 750 mg/kg/day for parental, reproduction and developmental toxicity.

Executive summary:

The reproductive toxicity of the test material was determined in accordance with the standardised guidelines OECD 422 and US EPA OPPTS 870.3650 under GLP conditions in a combined 28-day oral (gavage) repeated dose toxicity study with the reproduction/developmental toxicity screening test in the rat.

The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objectives of this study were to evaluate the potential toxic effects of the test material when administered for a minimum of 28 days to rats, and the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development.

The test material was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 750 mg/kg/day (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, water for injection, alone. Formulations were analysed once during the study to assess accuracy and homogeneity.

Males were treated for 30 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females that delivered were treated for at least 51 days, i.e. during 2 weeks prior to mating, during mating, during pregnancy, and during 13 days of lactation. Females which failed to deliver healthy offspring were treated for at least 41 days.

The following observations and examinations were evaluated: mortality / morbidity, clinical signs, functional observations and locomotor activity, body weight, food consumption, oestrous cycle determination, clinical pathology, measurement of thyroid hormone T4, macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: copulation and fertility indices, pre-coital time, number of implantation sites, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy).

There were no cases of test material-related mortality or adverse clinical signs in any group. There were no test material-related changes in parental body weight, food consumption, haematological and serum clinical chemistryanalyses[MD1] and there was no obvious effect of treatment on the auditory, pupillary and righting reflexes or gripping and open field tests for the males or females at any dose level.

There were no test material-related effects on mating performance of the males and females or on fertility in any group. There were 8, 8, 9 and 10 females that successfully delivered a litter in the control, 100, 300 and 750 mg/kg/day groups, respectively. There were no test material-related effects on the number of implantations, pre-birth loss, litter size, pup viability or body weight. There were no test material-related effects on areola/nipple retention. For the examined male pups, no nipples were observed at PND 13. Anogenital distance (normalised for body weight) in male and female pups was not affected by the treatment.

There was no evidence of any test material-related parental histological findings.

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material is at least 750 mg/kg/day for parental, reproduction and developmental toxicity.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

750 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available study was performed in accordance with standardised guidelines under GLP conditions. The quality of the database is considered to be high.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The reproductive toxicity of the test material was determined in accordance with the standardised guidelines OECD 421, OECD 422, US EPA OPPTS 870.3650 and US EPA OPPTS 870.3050 under GLP conditions in a combined 28-day oral (gavage) repeated dose toxicity study with the reproduction/developmental toxicity screening test in the rat.

The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objectives of this study were to evaluate the potential toxic effects of the test material when exposed for a minimum of 28 days and the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development.

The test material was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 750 mg/kg/day (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, water for injection, alone. Formulations were analysed once during the study to assess accuracy and homogeneity.

Males were treated for 30 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females that delivered were treated for at least 51 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 13 days of lactation. Females which failed to deliver healthy offspring were treated for at least 41 days.

The following observations and examinations were evaluated: mortality / morbidity, clinical signs, functional observations and locomotor activity, body weight, food consumption, oestrous cycle determination, clinical pathology, measurement of thyroid hormone T4, macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: copulation and fertility indices, pre-coital time, number of implantation sites, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy).

There were no cases of test material-related mortality or adverse clinical signs in any group. There were no test material-related changes in parental body weight, food consumption, haematological and serum clinical chemistry analyses and there was no obvious effect of treatment on the auditory, pupillary and righting reflexes or gripping and open field tests for the males or females at any dose level.

There were no test material-related effects on mating performance of the males and females or on fertility in any group. There were 8, 8, 9 and 10 females that successfully completed delivery in the control, 100, 300 and 750 mg/kg/day groups, respectively. There were no test material-related effects on the number of implantations, pre-birth loss, litter size, pup viability or body weight. There were no test material-related effects on areola/nipple retention. For the examined male pups, no nipples were observed at PND 13. Anogenital distance (normalised for body weight) in male and female pups was not affected by the test material.

There was no evidence of any test material-related parental histological findings.

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material is at least 750 mg/kg/day for parental, reproduction and developmental toxicity.

Effects on developmental toxicity

Description of key information

Screening: Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material is at least 750 mg/kg/day for parental and developmental toxicity.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Remarks:

screening study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 October 2016 to 08 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI (Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Males approximately 11 weeks and females approximately 13 weeks
- Weight at study initiation: males 344 to 411 g and females 209 to 276 g
- Fasting period before study: No
- Housing: Animals were housed in one air-conditioned room in a barrier protected unit. Males were housed in groups of 5 during the pre-mating and gestation periods. Females were housed in groups of 5 during the pre-mating period. Males and females were housed together during mating (one per sex per cage) and subsequently females were housed individually during gestation and with their litter during lactation.
Animals were housed in plastic cages, in compliance with European Regulations. Cellulose bedding or dust-free sawdust made from spruce tree wood was provided. A small amount (handful) of shredded paper was provided as enrichment for all animals. Any isolated animals had free access to a wooden gnaw block. Furthermore, tissues were also provided as enrichment for females towards the end of gestation.
- Diet: Rat pelleted complete diet ad libitum
- Water: Softened and filtered (0.2 μm) mains drinking water was available ad libitum (via an automatic watering system or bottles).
- Acclimation period: 8 days between animal arrival and start of treatment (males) or start of pre-test oestrous cycle smears (females).

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: > 35 %
- Air changes: At least 10 air changes per hour
- Photoperiod: 12 hours of light (artificial)/12 hours of darkness

IN-LIFE DATES
- From: Not specified
- To: 08 February 2017

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Water for injection

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was prepared as a suspension in the vehicle (water for injection). The test material was prepared at least once weekly and stored at 2 to 8 °C between uses. Formulations were maintained under continuous stirring for at least 15 minutes before and throughout the dosing procedure.

DOSE VOLUME: 10 mL/kg/day

VEHICLE
- Concentration in vehicle: 10, 30 and 75 mg/mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

STABILITY
Suspensions at 1 and 200 mg/mL were demonstrated to be stable for 8 days when stored refrigerated (+2 to +8 °C), based on the results of a study at the testing facility.

ANALYSIS OF PREPARATIONS
Quadruple (4 x 1 g) accurate samples were taken from each formulation, including the vehicle, used on the first day of treatment of the main study phase. Samples were taken from the top, middle and bottom levels and analysed for accuracy. Groups 2 and 4 were also assessed for homogeneity. One set of samples (2 x 1 g) was analysed at the testing facility and the other set (2 x 1 g) was stored frozen at -25 to -15 °C.

- Method
UPLC (pump, autosampler, detector): Acquity HClass , Waters
Column: Waters, Sunfire C18, 50 mm x 4.6 mm x 2.5 µm, reference 186003417
Precolumn: Not applicable
Pre-injection volume: Not applicable
Oven temperature: 30 °C
Injection volume: 10 µL
Run time: 9 min
Autosampler temperature: 20 °C
UV Detection: 210 ± 4.8 nm
Retention time: around 4.1 minutes
Flow rate: 1 mL/minute
Mobile Phase A: 0.2 % formic acid in water
Mobile Phase B: 0.2 % formic acid in acetonitrile
Elution gradient: At 0 and 2 minutes, 60 % A, 40 % B; at 7 and 7.1 minutes, 40 % A, 60 % B; at 9 minutes, 40 % A, 60 % B
Wash: 50/50 CH3CN/H2O
Purge/SW: 50/50 CH3CN/H2O
Correction factor for test material: 100 %

- Calibration range and Quality Control (QC)
16, 25, 40, 60 and 80 µg/mL prepared from two independent standard solutions. Standard solution was prepared at 200 µg/mL in dilution solvent (50/50 Acetonitrile/DMSO). Dilution factors were 2/25, 1/8, 1/5, 3/10 and 2/5.
The formulation concentration range was validated at 1 to 200 mg/mL in water for injection.

VALIDATION
The method was validated by the testing facility and a pass was achieved for the following method conformities:
- Quadratic regression of the calibration curves (r ≥0.999);
- Standard solution conformity between calibration and QC stock solutions (± 2 %);
- Analysis conformity: of calibration solutions (SE) recovery within 95-105 % of the exact concentrations (except for LLOQ samples 90-110 %);
- QC solutions: recovery within 95-105 % of the exact concentrations (except for LLOQ QC samples 90-110 %). One QC sample may be outside the acceptance criteria.

FORMULAE
- Quadratic regression of the calibration curves:
y = Cx2 + Bx + A
Where:
y = test material peak area (µAUs)
x = concentrations of the test material calibration solution (µg/mL)

- Back calculated concentration (µg/mL):
[-B + √(B2 - 4C(A-y ))] / 2C

- Standard solution conformity: Deviation between the Calibration standard solutions (SS Cal) and Quality Control standard solutions (SS QC)
Deviation (%) = [(SS Cal area / SS QC area) - 1] x 100

- Analysis conformity, the recovery of the back-calculated concentration of SE or SQC:
Recovery (%) = (Individual back calculated concentration / nominal concentration) x 100

- Concentrations of the formulation samples (suspension):
Concentration (mg/mL) = (Back calculated conc x V x F x d) / (sample weight (g) x 1000)
Where:
V = volume of the volumetric flask used
d = formulation density
F = dilution factor

- Deviation from the nominal concentration of the formulations:
Deviation (%) = [(mean of experimental concentration - nominal concentration) / nominal concentration] x 100

RESULTS
The acceptance range for achieved concentrations was ± 15 % from the nominal concentration for formulations at 10, 30 and 75 mg/mL.
All formulations at 10, 30 and 75 mg/mL in vehicle (water for injection) including the vehicle, used on the first day of treatment of the main study phase, were in agreement with acceptance criteria. The formulations at 10 and 75 mg/mL tested for homogeneity were homogenous. The deviations from the nominal concentrations ranged from -9.8 % to -1.9 % and the RSD was ≤2.3 %. No significant amount of test material was detected in the vehicle sample.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: After 2 weeks of treatment, animals were paired on the basis of one male and one female from the same group for a maximum of 14 days.
- Proof of pregnancy: The day of mating was confirmed by the presence of sperm in a vaginal smear and was recorded and taken as day 0 of gestation (G 0).
- After successful mating each pregnant female was caged (how): Mated females were separated from the males once mating had been confirmed and smearing ceased and housed individually.

Duration of treatment / exposure:

Females that delivered were treated for at least 51 days, i.e. during 14 days prior to mating (the first day of dosing is designated as Day 1), the variable time to conception (the first day of gestation is designated as G 0), the duration of the pregnancy and 13 days after delivery (the first day of birth is designated as L 0), up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for at least 41 days.

Frequency of treatment:

Once daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

750 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Rationale for the dose selection was based on the results of the range-finder in which administration of the test material at doses of 500 and 1000 mg/kg/day was associated with a minimal effect on body weight gain in the high dose group with no obvious correlation effect on food consumption. For the main study phase, a high dose of 750 instead of 1000 mg/kg/day was employed to limit maternal toxicity since in the main phase, the animals were pregnant and assumed to be likely more sensitive than non-pregnant animals. Moreover, the duration of the treatment was defined to be at least 51 days (depending on the duration of the mating period).
- Animal assignment: Performed during the acclimatisation period, using a computer-generated randomisation. The mean body weights of each group at allocation were not statistically significantly different from each other (analysis of variance), each sex being considered separately.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All adults were observed twice daily, at the beginning and at the end of each working day (including weekends and public holidays) for morbidity/mortality. The animals were observed daily for clinical signs. To detect any clinical signs or reactions to treatment, the animals were observed before and once after dosing (based on information provided from the range-finder).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A full clinical examination was performed on each weighing day. Towards the end of the gestation, females were examined daily for signs of parturition.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed at the time of randomisation, on the first day of exposure (prior to the first exposure) and weekly thereafter during pre-mating and mating periods. Mated females were weighed on days 0, 4, 7, 11, 14, 17 and 20 of gestation and on days 1, 4, 7 and 13 of lactation.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption of females was recorded for the following periods: weekly during the pre-mating period, days 0 to 4, 4 to 7, 7 to 11, 11 to 14, 14 to 17 and 17 to 20 of gestation and days 1 to 4, 4 to 7 and 7 to 13 of lactation.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the pre- mating period (Day 14) blood was withdrawn from the retro-orbital sinus. Samples were collected in K2-EDTA tubes for haematology parameters and in tubes containing trisodium citrate for coagulation parameters.

- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes, for about 17 hours
- How many animals: First 5 females/group
- Parameters examined for haematology: Haemoglobin, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, packed cell volume, red blood cell count, mean corpuscular volume, reticulocyte count, platelet count, total white blood cell count and differential white blood cell count.
-Parameters examined for coagulation: Prothrombin time, activated partial thromboplastin time and fibrinogen.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the pre- mating period (Day 14) blood was withdrawn from the retro-orbital sinus. Samples were collected in tubes without anticoagulant.
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes, for about 17 hours
- How many animals: First 5 females/group
- Parameters examined: Sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, total cholesterol, total bilirubin, total protein, albumin, creatinine, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase and bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Females were examined on PND 12 (penultimate day of dosing) or PND 13 (last day of dosing), 2 or 1 day(s) before scheduled necropsy, respectively.
- Dose groups that were examined: 5 selected animals per group (females selected had live pups); the same animals were selected for terminal investigations.
- Battery of functions tested: Auditory reflex, pupillary reflex, righting reflex, fore- and hind-limb grip strength and locomotor activity in an open field test.
In the locomotor activity in an open field test, activity was monitored by a video image analysis system (Videotrack supplied by Viewpoint, Lyon, France). The arena is divided into nine equal invisible regions; the time spent by the animal in each type of region (i.e. corner, centre or lateral) was recorded. Motor activity is divided into three categories: ambulatory activity (in which the centre of the image moves at more than 7 cm/sec), small movements (including grooming etc.) and inactivity. The proportion of time spent engaged in each type of activity and the total distance travelled by the rat were calculated.

IMMUNOLOGY: No

HORMONE ANALYSIS: Yes
- Time schedule for collection of blood: Terminal blood sampling. For F0-females on lactation day 14; for unmated F0-females 28 days after the last day of the mating period; and F0-females (non-pregnant and the one with total litter death) on Day 26 of gestation. Blood samples of approximately 0.9 mL (target volume) were withdrawn from a retro-orbital sinus. Samples were collected in tubes without anticoagulant, between 7:00 and 9:00 a.m. because of diurnal variation of hormone concentrations. The samples were centrifuged at 1800 g, at approximately 4 °C for 10 minutes and the serum was separated in two aliquots (at least 150 μL for the first aliquot and the remainder for the second aliquot). Serum samples were stored deep-frozen (between -70 and -90 °C) and allowed to thaw prior to analysis.
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: No
- How many animals: All females (except group 3 female no. 152 which was found dead on Day 14) on the day of necropsy.
- Parameters examined: T4 and TSH levels were examined if required.

OESTRUS CYCLES: Yes
- Time schedule for examinations: Daily vaginal smears were performed to determine the stage of oestrus beginning 14 days prior to treatment, the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal smears continued for any female with no evidence of copulation until termination of the mating period. On the day of scheduled necropsy, a vaginal smear was taken to determine the stage of the oestrous cycle and allow correlation with histopathology of female reproductive organs.

PREGNANCY AND PARTURITION: Yes
- Time schedule for examinations: The following was recorded for each female: date of mating, date of parturition (day 0 of lactation or L 0), duration of gestation and abnormalities of nesting or nursing behaviour.

GROSS PATHOLOGY: Yes
Surviving adult animals were killed by carbon dioxide inhalation followed by exsanguination. Group 1 female no. 111 with total litter death was killed by carbon dioxide inhalation and exsanguination then necropsied. Group 3 female no. 152 found dead on Day 14 was also necropsied. Any such females found after the first day of pairing were given a caesarean section to determine pregnancy status, number of corpora lutea and numbers and types of uterine implantations.
All animals were submitted to a macroscopic examination for structural or pathological changes, with special attention being paid to the reproductive organs. The uterus of all adult females was placed in ammonium sulphide solution in order to confirm the number of implantation sites.
Animals which delivered were subjected to necropsy on day 14 of lactation, females which failed to deliver on post-coitum Day 26 (females with evidence of mating) or 28 days after the last day of the mating period (female without evidence of mating). For the female with total litter loss, necropsy took place within 24 hours of litter loss. In the case of spontaneous death, necropsy took place on the day of death.
Five animals per group were subjected to full macroscopic examination and recording of organ weights. The females selected all had live pups.
The following organ weights were recorded for the selected animals: Adrenal glands, brain, cervix and uterus, heart, kidneys, liver, ovaries, parathyroid and thyroid glands, spleen and thymus. For the other animals of each group, ovaries, thyroid and uterus were weighed. Paired organs were weighed together. The organs were weighed after dissection of fat and other contiguous tissues.

HISTOPATHOLOGY: Yes
All organs/tissues listed below were sampled for the 5 randomly selected adult females of each group (for females, only those with live pups) and for group 3 female no. 152 found dead on Day 14. Only a limited list of organs/tissues was sampled for all remaining animals (including females that failed to produce a viable litter by day 26 post coitum, or with total litter death).
All organs/tissues sampled were fixed and preserved in 10 % neutral formalin with the exception of Harderian glands, eyes and optic nerves which were fixed in modified Davidson's fluid. The same sampling and trimming procedures were used for all applicable tissues in all applicable dose groups. Blocks were prepared only for tissues which were evaluated histopathologically.
Histopathological examinations were performed as follows:
- For all organs/tissues from group 3 female no. 152 found dead on Day 14
- For all organs/tissues from the 5 randomly selected adult females of groups 1 (control) and 4 (high dose)
- For the reproductive organs of all females that failed to deliver healthy pups
Organs and tissues examined were: macroscopic lesions, adrenal glands, bone (femur) and articulation, bone (sternum) with bone marrow, brain, caecum, cervix, clitoral gland, colon, duodenum, eyes, Harderian glands, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (mandibular and mesenteric), mammary gland (inguinal region with skin), oesophagus, optic nerves, ovaries, parathyroid glands, Peyer’s patches, pituitary gland, rectum, sciatic nerve (left), seminal vesicles, skeletal muscle, skin, spinal cord (cervical thoracic and lumbar), spleen, stomach, thymus, thyroid glands, trachea, urinary bladder, uterus and vagina.
All sections were stained with haematoxylin and eosin.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes

All animals were submitted to a macroscopic examination for structural or pathological changes, with special attention being paid to the reproductive organs. The uterus of all adult females was placed in ammonium sulphide solution in order to confirm the number of implantation sites.

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
The size of each litter was adjusted to 8 pups on PND 4 by eliminating extra pups by random selection to yield 4 male and 4 female pups per litter where possible. Blood samples were collected from two surplus pups where possible, pooled and used for determination of serum T4 levels. No pups were eliminated when litter size dropped below the culling target (8 pups/litter). When there was only one pup available above the culling target, only one pup was eliminated and used for blood collection for possible serum T4 assessments.

PARAMETERS EXAMINED
-The following were recorded for each F1 litter: number of pups born (live and dead); external abnormalities of the pups; number, weight and sex of pups alive on PND’s 1, 4, 7 and 13; anogenital distance (normalised to the cube root of body weight) on PND 1; and presence of areola/nipples on PND 13 for all males in each litter.

GROSS EXAMINATION OF DEAD PUPS: Yes
Pups that died were necropsied. Any external abnormalities observed were recorded. Their stomach was examined for the presence of milk, if possible. Defects or cause of death was evaluated, if possible.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

HORMONE ANALYSIS: Yes
- Time schedule for collection of blood: Terminal blood sampling.
On PND 4, blood samples of approximately 0.4 mL (target volume) were obtained by decapitation under anaesthesia. Blood samples from the pups per litter were collected into one serum tube. When the target volume of 0.4 mL could not be reach by pooling 2 pups, extra pups were sampled if available. When only 1 surplus pup per litter was available at culling, as much blood as possible was collected from this single pup.
On PND 13, blood samples of approximately 0.9 mL (target volume) were withdrawn by intracardiac puncture under anaesthesia.
Samples were collected in tubes without anticoagulant, between 8:00 and 10:30 a.m. because of diurnal variation of hormone concentrations. The samples were centrifuged at 1800 g, at approximately 4 °C for 10 minutes and the serum was separated into an aliquot of at least 150 μL on PND 4 and into two aliquots (at least 150 μL for the first aliquot and the remainder for the second aliquot) on PND 13.
Serum samples were stored deep-frozen (between -70 and -90 °C) and allowed to thaw prior to analysis.
- Anaesthetic used for blood collection: Yes, sodium pentobarbitone
- Animals fasted: No
- How many animals: For 2 pups per litter where possible on PND4 and two pups per litter where possible on PND 13 (one male and one female on each occasion where possible).
- Parameters examined: T4 was examined in all PND 13 pups and PND 4 pups if required. TSH levels were measured in PND13 pups if required.

GROSS PATHOLOGY: Yes
On PND 4 and PND 13, pups were killed by an intraperitoneal injection of sodium pentobarbitone. Pups culled were necropsied on PND 4. Pups surviving to terminal sacrifice were necropsied on PND 13.
Each pup was sexed and examined for external defects with special attention being paid to the external reproductive organs. The thyroid glands of one male and one female by litter were fixed and preserved in 10 % formalin. Any external abnormalities observed were recorded but not preserved.

HISTOPATHOLOGY: No

Statistics:

Analyses were performed by the Provantis acquisition system. The best transformation for the data (none, log or rank) was determined depending upon the kurtosis of the data and the probability of the Bartlett's test for homogeneity of the variances and an assessment of whether the size of the groups were approximately equal or not.
Non- or log-transformed data were analysed by parametric methods. Rank transformed data were analysed using non-parametric methods.
Data were then analysed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non-parametric data.
If no trend was found and means were not homogeneous, the data were analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
Selected incidence data were analysed using the Provantis data acquisition system and/or an SAS software package. A chi2 test was used for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.
The locomotor activity in an open field and pre-coital interval data were analysed using an SAS software package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk's test was used to assess the normality of the data distribution in each group. Data with homogenous variances and normal distribution in all groups were analysed using ANOVA followed by Dunnett’s test. Data showing non-homogenous variances or a non-normal distribution in at least one group were analysed using Kruskal-Wallis test followed by the Wilcoxon's rank sum test.
Microsoft Excel® (version 2003 or higher) was employed to present certain results and perform associated calculations.

Indices:

Pre-coital interval (in days): sum of days until successful insemination / number of inseminated females
Male and female copulation index (in %): (number of inseminated females / number of paired animals) x 100
Male and female fertility index (in %): (number of pregnant females / number of inseminated females) x 100
Pre-birth loss (in %): [(number of implantations - number of offspring born) / number of implantations] x 100
Live birth index (in %): (number of pups born alive / number of pups born) x 100
Viability index (in %): (number of pups alive on PND 4 / number of pups alive at birth) x 100
Lactation index (in %): (number of pups alive on PND 13 / number of pups alive on PND 4 (after culling)) x 100
Sex ratio (proportion of male pups in %): (number of males / number of pups) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related clinical signs in any group. Yellow-coloured faeces were noted in treated groups at 300 and 750 mg/kg/day. This was attributed to the yellow colour of the test material. Control female no. 113 had marked piloerection associated with decreased activity on lactation Day 1. These signs were most probably due to parturition and were no longer observed thereafter. Other incidental clinical observations included localised hairloss, chromodacryorrhea and/or scabs.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no test material-related deaths in any group. Control female no. 111 was euthanised on gestation Day 26 due to total litter resorption. Female no. 152 given 300 mg/kg/day was found dead on Day 14 during the pre-mating period following anaesthesia for blood sampling. In view of their nature and incidence, these isolated deaths were considered incidental.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes in body weights or body weight gains that were considered to be related to the test material during the pre-mating, gestation or lactation periods. Any differences noted in mean body weight gains were incidental since they were either not dose-related, transient or since mean values in treated groups were superior to those in the concurrent controls.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically-relevant changes in food consumption during the pre-mating, gestation or lactation periods. Mean food consumption was slightly lower in treated than in control females during the pre-mating period (up to -4.8 % in the 750 mg/kg/day group). Since this change was transient and not observed during the gestation or lactation periods and had no adverse effect on body weights, it was not considered toxicologically relevant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences noted in haematological and coagulation parameters between control and treated rats that were considered to be toxicologically relevant.
Although differences in mean values attained statistical significance for females compared with the concurrent controls, the decrease in mean white blood cell and lymphocyte counts was of no toxicological significance in view of its low magnitude. In addition, mean values were within the historical control range.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences noted in serum clinical chemistry parameters between control and treated rats that were considered to be toxicologically relevant.
Statistically significant dose-related slightly decreased mean sodium concentration was noted on Day 14. All mean values remained, however, within the historical control ranges. These findings were therefore of no toxicological relevance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed.
Fore- and hind-limb grip strength mean values at 750 mg/kg/day were higher than controls for treated females. This was considered to be due to intra-group variability and therefore incidental.
The variation in motor activity (open field) did not indicate a relation with treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

When compared with controls, there were no test material-related changes in organ weights associated with the administration of the test material.
Occasional organ weight differences were considered to be result of the terminal body weight differences or only to reflect normal individual variation and unrelated to test material.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no observations that were considered to be associated with administration of the test material. Occasional gross lesions were noted with variable incidence in controls and treated animals and were considered to be incidental. Most of these findings were histologically correlated and considered to be part of the spontaneous background observations normally encountered in the rodents.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There was no evidence of any test material-related microscopic findings in animals given 750 mg/kg/day.
The nature or incidence of histological findings in the organs examined did not indicate any relationship to treatment and they were considered to be incidental and part of the normal background changes normally encountered in reproductive/developmental studies in rodents.

- Premature Decedents
Female no. 152 given 300 mg/kg/day was found dead on Day 14 during the pre-mating period following isoflurane anaesthesia for blood sampling. At the necropsy, a bilateral dilated kidney was noted, correlating histologically with slight pelvic dilatation, considered to be incidental and unrelated to treatment.
Control female no. 111 was euthanised due to total litter resorption. Histological evaluation of genital tract showed a normal oestrous cycle.

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

The percentage of pre-birth loss was not affected by treatment.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean number of implantation sites was comparable in all groups. The percentage of pre-birth loss was not affected by treatment.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no test material-related effects on pup viability.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

The mean duration of gestation was comparable in all groups.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All surviving animals mated with the exception of one pair in the 100 mg/kg/day group. Most mated females showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal oestrous cycle), with the exception of one female in each of the control (no. 118) and 300 mg/kg/day (no. 154) groups with a pre-coital interval of 9 and 14 days, respectively. The mean pre-coital interval was therefore slightly higher in these two groups but this was considered incidental.
There was no effect of treatment on fertility of either sex in any group. There was one female in each of the control and 100 mg/kg/day groups (nos. 118 and 136, respectively) that did not become pregnant. These isolated cases, including controls, were considered incidental.
There were 8, 8, 9 and 10 females that successfully completed delivery in the control, 100, 300 and 750 mg/kg/day groups, respectively.

Other effects:

no effects observed

Description (incidence and severity):

There was no test material-related influence on mating performance of the males and females in any group.
Length and regularity of the oestrous cycle were not affected by treatment with the test material.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 750 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No adverse effects were observed that were attributed to treatment with the test material

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no obvious test material-related changes in pup body weights
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There were no obvious test material-related changes in pup body weights. The mean pup body weights (males and females) were slightly lower in the 300 and 750 mg/kg/day groups than in the control and 100 mg/kg/day groups from days 1 to 13 of lactation. These minor differences were considered to be due to the slightly higher live litter size at birth in the treated groups and not to treatment with test material.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related differences in pup viability. The number of offspring born alive compared to the total number of offspring born was not affected by treatment. There was one pup dead or missing in the control group versus none in the treated groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The mean percentage of males per litter was abnormally low in the control group (30.8 %). This was considered incidental.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related differences in litter size. The mean litter size at birth was higher in the 300 and 750 mg/kg/day groups than in the control and 100 mg/kg/day groups. This was considered incidental.

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on Day 4 before culling compared to the number of offspring alive at birth was not affected by treatment. Two, three and two pups in the control, 300 and 750 mg/kg/day groups were found dead or missing versus none in the 100 mg/kg/day group between postnatal Days 0 and 4.
The number of live offspring on Day 13 compared to the number of offspring on Day 4 (post culling) was not affected by treatment. There was one pup found dead or missing in the 300 mg/kg/day group versus none in the other groups.
No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

External malformations:

no effects observed

Description (incidence and severity):

There were no gross observations that were considered to be associated with administration of the test material.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There were no pup observations that suggested any association with maternal treatment. Incidental occasional findings included weak or thin pups, incomplete hair growth or haematoma.

T4 HORMONE ANALYSIS
No differences in Total T4 levels were noted among the different groups of PND 13 pups.

MALE PUPS AREOLA/NIPPLES
There were no test material-related effects on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

ANOGENITAL DISTANCE
Anogenital distance (normalised for body weight) in male and female pups was not affected by treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed that were attributed to treatment with the test material

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material is at least 750 mg/kg/day for parental, reproduction and developmental toxicity.

Executive summary:

The developmental toxicity of the test material was determined in accordance with the standardised guidelines OECD 421, OECD 422, US EPA OPPTS 870.3650 and US EPA OPPTS 870.3050 under GLP conditions in a combined 28-day oral (gavage) repeated dose toxicity study with the reproduction/developmental toxicity screening test in the rat.

The objectives of this study were to evaluate the potential toxic effects of the test material when exposed for a minimum of 28 days and the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development.

The test material was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 750 mg/kg/day (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, water for injection, alone. Formulations were analysed once during the study to assess accuracy and homogeneity.

Males were treated for 30 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females that delivered were treated for at least 51 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 13 days of lactation. Females which failed to deliver healthy offspring were treated for at least 41 days.

The following observations and examinations were evaluated: mortality / morbidity, clinical signs, functional observations and locomotor activity, body weight, food consumption, oestrous cycle determination, clinical pathology, measurement of thyroid hormone T4, macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: copulation and fertility indices, pre-coital time, number of implantation sites, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy).

There were no cases of test material-related mortality or adverse clinical signs in any group. There were no test material-related changes in parental body weight, food consumption, haematological and serum clinical chemistry analyses and there was no obvious effect of treatment on the auditory, pupillary and righting reflexes or gripping and open field tests for the males or females at any dose level.

There were no test material-related effects on mating performance of the males and females or on fertility in any group. There were 8, 8, 9 and 10 females that successfully completed delivery in the control, 100, 300 and 750 mg/kg/day groups, respectively. There were no test material-related effects on the number of implantations, pre-birth loss, litter size, pup viability or body weight. There were no test material-related effects on areola/nipple retention. For the examined male pups, no nipples were observed at PND 13. Anogenital distance (normalised for body weight) in male and female pups was not affected by the test material.

There was no evidence of any test material-related parental histological findings.

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material is at least 750 mg/kg/day for parental, reproduction and developmental toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

750 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available study was performed in accordance with standardised guidelines under GLP conditions. The quality of the database is considered to be high.

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

The developmental toxicity of the test material was determined in accordance with the standardised guidelines OECD 422 and US EPA OPPTS 870.3650 under GLP conditions in a combined 28-day oral (gavage) repeated dose toxicity study with the reproduction/developmental toxicity screening test in the rat. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objectives of this study were to evaluate the potential toxic effects of the test material when administered to rats for a minimum of 28 day,s and the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development.

The test material was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 750 mg/kg/day (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, water for injection, alone. Formulations were analysed once during the study to assess accuracy and homogeneity.

Males were treated for 30 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females that delivered were treated for at least 51 days, i.e. during 2 weeks prior to mating, during mating, during gestation, and during 13 days of lactation. Females which failed to deliver healthy offspring were treated for at least 41 days.

The following observations and examinations were evaluated: mortality / morbidity, clinical signs, functional observations and locomotor activity, body weight, food consumption, oestrous cycle determination, clinical pathology, measurement of thyroid hormone T4, macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: copulation and fertility indices, pre-coital time, number of implantation sites, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy).

There were no cases of test material-related mortality or adverse clinical signs in any group. There were no test material-related changes in parental body weight, food consumption, haematological and serum clinical chemistry analyses and there was no obvious effect of treatment on the auditory, pupillary and righting reflexes or gripping and open field tests for the males or females at any dose level.

There were no test material-related effects on mating performance of the males and females or on fertility in any group. There were 8, 8, 9 and 10 females that successfully completed delivery in the control, 100, 300 and 750 mg/kg/day groups, respectively. There were no test material-related effects on the number of implantations, pre-birth loss, litter size, pup viability or body weight. There were no test material-related effects on areola/nipple retention. For the examined male pups, no nipples were observed at PND 13. Anogenital distance (normalised for body weight) in male and female pups was not affected by the test material.

There was no evidence of any test material-related parental histological findings.

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material is at least 750 mg/kg/day for parental, reproduction and developmental toxicity.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to reproductive and developmental toxicity.