(3AS,4S,6AR)-4-METHOXYTETRAHYDROFURO[3,4- B]FURAN-2(3H)-ONE

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-12-28 to 2017-03-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: U.S. EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

443-930-6

EC Name:

-

Cas Number:

866594-60-7

Molecular formula:

C7H10O4

IUPAC Name:

(3AS,4S,6AR)-4-METHOXYTETRAHYDROFURO[3,4- B]FURAN-2(3H)-ONE

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M16CB3356
- Expiration date of the lot/batch: 2018-03-12
- Purity test date: 2016-03-12

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Stability for at least 6 hours at room temperature was confirmed over the concentration range 1 to 200 mg/mL as part of the analytical method development and validation study (Test Facility Study No. 514723).

FORM AS APPLIED IN THE TEST (if different from that of starting mateiral): suspension

OTHER SPECIFICS: correction factor is 1

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males approx. 10 weeks (at start F0-treatment); females approx. 11 weeks (at start pretest) and approx. 13 weeks (at start F0-treatment).
- Weight at study initiation: Males: 274-305 g; Females: 211-247 g
- Fasting period before study: no
- Housing:
Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage.Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet.
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): at least 10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES:
From: 2016-12-28 (start pretest); 2017-01-11 (start treatment); 2017-02-17/18/19/20/21 and 2017-03-03 (delivery of litters)
To: 2017-02-09 (necropsy males); 2017-03-01/02/05/06/16 (necropsy pups); 2017-03-02/03/06/07/17 (necropsy females)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

, specific gravity 1.036

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test item. Adjustment was made for specific gravity of the vehicle. A correction was made for the purity/composition of the test item. A correction factor of 1 was used. Formulations were placed on a magnetic stirrer during dosing.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch.
- Concentration in vehicle: 0 mg/mL (group 1), 22 mg/mL (group 2), 66 mg/mL (group 3), 200 mg/mL (group 4)
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
- Lot/batch no. (if required): no data
- Purity: no data

Details on mating procedure:

- M/F ratio per cage: 1:1 basis
- Length of cohabitation: Following a minimum of 14 days of treatment for the males and females, until detection of mating was confirmed.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating was confirmed, the males and females were separated.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (17 January 2017, Day 7 of treatment) according to a validated method (Test Facility Study No. 514723). Three sets of duplicate samples were collected. Two sets of duplicate samples were stored in the refrigerator as reserve samples. Once analytical results were approved in the raw data by the Principal Scientist, the reserve samples were destroyed. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
In addition to the criteria mentioned in the validated analytical method, each calibration curve was accepted if the average of the retention times and response factors of the data points used to construct the calibration line were within a range of ±10.00% compared to those obtained during the method validation.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85.00-115.00% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10.00%. Formulations were considered stable if the relative difference before and after storage was maximally 10.00%.
Stability of formulations over 6 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/mL) was determined as part of the analytical method development and validation study (Test Facility Study No. 514723).

Duration of treatment / exposure:

29 days (males), 50-65 days (females that delivered). Female nos.59 (Group 2), 61 (Group 3) and 71 and 74 (Group 4) were left out from treatment for one day as they were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation. Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (Control group)

Doses / concentrations 2

Dose / conc.:

110 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

330 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on the results of a subchronic 28-day toxicity study, in which dose levels of 0, 50, 200 and 1000 mg/kg/day were tested. No test item related toxicity was observed up to 1000 mg/kg/day.
- Rationale for animal assignment (if not random): Randomized

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals.

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. Relative food consumption was calculated and reported.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: not applicable.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Limited to thyroid hormone analysis.
- End of study from all animals at planned necropsy; this included females on Day 14-16 of lactation, non-pregnant females and all males after at least 4 weeks of treatment.
- Blood samples were collected under anaesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of approximately 24 hours) before blood sampling, but water was available. Blood samples (0.9 mL) were drawn from the retro-orbital sinus and collected into serum tubes.
- Males: 1 aliquot of 150 μL serum was used for measurement of thyroxine (T4), and the remaining volume of serum was stored for possible future measurement of thyroid stimulating hormone (TSH).
- Females: The serum was stored for possible future measurement of thyroxine (T4) and/or thyroid stimulating hormone (TSH).

Oestrous cyclicity (parental animals):

Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period. During pretest, this was done for 48 females. On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.

Sperm parameters (parental animals):

Parameters examined in F0 male parental generation: additional slides of the testes to examine staging of spermatogenesis; testis weight.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No. All pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink on post-natal day 1 (= day the litter was found completed).
- To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups. Selective elimination of pups, e.g. based upon body weight, or anogenital distance was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in F1:
- Mortality/viability: The numbers of live and dead pups were determined on PND 1 and daily thereafter. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check were reported in the respective table of the study report.
- Body weights: Live pups were weighed on PND 1, 4, 7 and 13.
- Sex: Sex was determined for all pups on PND 1 and 4.
- Anogenital distance: Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
- Areola/nipple retention: On PND 13, all males in each litter were examined for the number of areola/nipples.

GROSS EXAMINATION OF DEAD PUPS:
All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, following completion of the mating period (a minimum of 28 days of dose administration).
- Maternal animals: All surviving animals, on PND 14-16 (females which delivered). on days 25 or 26 (females which failed to deliver, with evidence of mating).
- Spontaneous deaths: As soon as possible after death and always within 24 hours (Female No. 44).

GROSS NECROPSY
- All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated. After sacrifice, all animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- The number of former implantation sites were recorded for all paired females.
- For females which failed to deliver a complete nest, uterine contents (i.e. any fetuses, placenta and implantation sites) were fixed (if applicable), but was not examined histopathologically.
- Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution): Cervix (F), Coagulation gland (M), Epididymides (M), Mammary gland area inguinal region with skin (M/F), Ovaries (F), Pituitary gland (M/F), Prostate gland (M), Seminal vesicles (M), Testes (M), Thyroid including parathyroid if detectable (M/F), Uterus (F), Vagina (F), All gross lesions (M/F).

ORGAN WEIGHTS
- Absolute organ weights and organ to body weight ratios were reported.
- The following organ weights and terminal body weight were recorded from all animals on the scheduled day of necropsy: Epididymides, Prostate gland, Seminal vesicles (including coagulation gland), Testes, Thyroid.

HISTOPATHOLOGY
- All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin.
- The following slides were examined by a pathologist: The ovaries, testes, epididymides and thyroids of the animals of Groups 1 and 4; Additional slides of the testes of all males of Groups 1 and 4 and all males that failed to sire to examine staging of spermatogenesis; All gross lesions of all animals (all dose groups); The preserved organs and tissues of the female no. 44 which died spontaneously.
- All abnormalities were described and included in the study report. An attempt was made to correlate gross observations with microscopic findings.
- A peer review on the histopathology data was performed by a second pathologist.

Postmortem examinations (offspring):

SACRIFICE
- On PND 4 (at culling), 2 pups per litter were killed by decapitation for blood collection.
- On PND 13-15, 2 pups per litter were anaesthetized using isoflurane for blood collection by aorta puncture followed by exsanguination.
- All remaining pups (PND 13-15) were sacrificed using Euthasol 20% by intraperitoneal injection.

GROSS NECROPSY
- All pups were sexed both externally and internally. Descriptions of all abnormalities were recorded.
- At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood sampling, was preserved in 10% buffered formalin

HISTOPATHOLOGY / ORGAN WEIGTHS
not examined

Statistics:

The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

For each group, the following calculations were performed:
Mating index (%) = (Number of females mated/Number of females paired) x 100
Fertility index (%) = (Number of pregnant females/Number of females mated) x 100
Gestation index (%) = (Number of females bearing live pups/Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Survival indices:
Post-implantation survival index (%) = (Total number of offspring born/Total number of uterine implantation sites) x 100
- Post-implantation survival index was expressed as 100% when the number of offspring exceeded the number of implantation sites recorded.

Live birth index (%) = (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100

Viability index (%) = (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) x 100

Lactation index (%) = Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling)) x 100
- Group mean values were calculated from individual litter values.

Sex ratio (percentage males) = (Number of males in litter/Total number of offspring in litter) x 100

Percentage of live males at first litter check (%)

Percentage of live females at first litter check (%)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs of toxicity were noted during the observation period.

Salivation seen after dosing among animals of the treatment groups and one control female during the treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).

Incidental findings that were noted included piloerection, rales, chromodacryorrhoea, alopecia, scabs and scales swelling of the neck. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were not considered to be signs of toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.

One control female died on Day 1 of lactation, she had delivered a complete litter. The 15 pups of this litter had to be killed in extremis since they were cold and less milk was noted. No clinical signs or effects on body weight or food consumption were noted prior to the death of this female. At necropsy, beginning autolysis and black-brown contents of jejunum and caecum were observed. As this female was treated with vehicle, this was not related to treatment with the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gain of treated animals were unaffected by treatment up to 1000 mg/kg/day.

In the high dose females, body weights were statistically significantly lower on the first day of treatment (prior to dosing) and remained slightly lower compared to the concurrent controls throughout the study. Furthermore, body weight gain was normal in these females. Therefore, the slightly reduced body weights were considered a chance finding and not related to treatment with the test item.

The statistically significantly increased body weight gain on the first day of mating in females treated at 330 mg/kg/day was considered a chance finding.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after allowance for body weight were noted.

The slight increase in relative food consumption in the 1000 mg/kg/day treated females during lactation was within the normal range; it was considered of no toxicological relevance.

The slight increase in relative food consumption between Days 4 to 7 post-coitum was considered a chance finding.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum levels of T4 in F0 males were unaffected by treatment up to 1000 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations.

All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

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

Most females had regular cycles of 4 days. Extended di-estrus occurred in female no. 42 with a regular cycle. As this occurred in a female treated with vehicle only, this occurrence was not test item-related.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Spermatogenic staging profiles were normal for all males examined.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

REPRODUCTION DATA
- Mating index: Mating index was not considered to be affected by treatment. All females showed evidence of mating.
- Precoital time: Precoital time was considered not to be affected by treatment up to 1000 mg/kg/day.
- Number of implantation sites: Number of implantation sites was unaffected by treatment.
- Fertility index: Fertility was unaffected by treatment up to 1000 mg/kg/day. All mated females were pregnant.

DEVELOPMENTAL DATA
- Gestation index and duration: Gestation index and duration of gestation were considered not to be affected by treatment.
- Parturition/maternal care: No signs of difficult or prolonged parturition were noted among the pregnant females in the treatment groups. Female no. 44 (control group) was found dead on Day 1 of lactation after delivering a complete litter. It could not be excluded that she died due to complications following parturition; however, as it concerned a control female this was not related to treatment with the test item. No deficiencies in maternal care were observed.
- Post-implantation survival index: The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment. For female nos. 42 (control) and 76 (1000 mg/kg/day), the number of pups were slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation. No toxicological relevance was attached to this finding.
- Litter size: Litter size was not considered affected by treatment up to 1000 mg/kg/day.
- Live birth index: The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment. Two pups at 110 mg/kg/day and one pup in the 330 mg/kg/day group were found dead at first litter check. At the incidence observed and without a dose-related trend no toxicological relevance was attributed to these dead pups.
- Viability index: The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered affected by treatment.
Female no. 44 of the control group was found dead on Day 1 of lactation, her litter (15 pups) was subsequently sacrificed in extremis as they were cold and had less milk. The low viability index in the control group and statistically significantly lower postnatal loss in the treatment groups could be contributed to this individual litter in the control group. Five pups of the 110 mg/kg/day group and three pups at 330 mg/kg/day were found dead or were missing on Day 2 or 4. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
- Lactation index: The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was unaffected by treatment. No pups were found dead/missing between lactation Days 5 and 13.

Details on results (P0)

Parental results:
No parental toxicity was observed up to the highest dose level tested (1000 mg/kg/day). No toxicologically significant changes were noted in any of the parental parameters investigated in this study (i.e. mortality, clinical appearance, body weight & body weight gain, food consumption, T4 thyroid hormone analysis (males), macroscopic examination, organ weights and microscopic examination).

Reproductive results:
No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg/day). No treatment-related or toxicologically significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Analysis of dose preparations:
- Accuracy of preparation: The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85.00% and 115.00%). A small response at the retention time of the test item was observed in one of the chromatograms of the Group 1 formulation. It was considered not to derive from the formulation since the response was not observed in the duplicate test sample.
- Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10.00%).

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse changes were noted in any of the parameters evaluated in this study.

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse changes were noted in any of the parameters evaluated in this study.

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment. The nature and incidence of other clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered affected by treatment.

Female no. 44 of the control group was found dead on Day 1 of lactation, her litter (15 pups) was subsequently sacrificed in extremis as they were cold and had less milk. The low viability index in the control group and statistically significantly lower postnatal loss in the treatment groups could be contributed to this individual litter in the control group.

Five pups of the 110 mg/kg/day group and three pups at 330 mg/kg/day were found dead or were missing on Day 2 or 4. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 13-15 pups were considered not to be affected by treatment.

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):

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

The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

- Sex ratio: Sex ratio was considered not to be affected by treatment.
- Anogenital distance: Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.
The slight increase in mean anogenital distance observed in female pups of the 330 and 1000 mg/kg/day groups was not statistically significant and within normal limits and was therefore considered not to be toxicologically relevant.
- Areola/nipple retention - Treatment up to 1000 mg/kg/day had no effect on areola/nipple retention, nipples were not observed at PND 13 for any of the examined male pups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

DEVELOPMENTAL RESULTS
- No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg/day).
- No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance (PND 1), areola/nipple retention (PND 13 males), T4 thyroid hormone levels (PND 4 and 13-15) and macroscopy.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, treatment with JNJ-31064345-AAA (T002907) by oral gavage in male and female Wistar Han rats at dose levels of 110, 330 and 1000 mg/kg revealed no parental, reproduction or developmental toxicity up to 1000 mg/kg.
Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: at least 1000 mg/kg
Reproduction NOAEL: at least 1000 mg/kg
Developmental NOAEL: at least 1000 mg/kg
Therefore, the substance is not classified as a reproductive toxicant according to the CLP Regulation.