D-Glucitol, 1,5-anhydro-1-C-[4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl]-, 2,3,4,6-tetraacetate, (1S)-

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 February 2016 - 28 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same 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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other:

Principles of method if other than guideline:

In addition, the procedures described in this report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 2016);
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Develo pmental Toxicity Screening Test (July 2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)

GLP compliance:

yes (incl. QA statement)

Remarks:

d.d. 3 November 2015

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

[(2R,3R,4R,5S,6S)-3,4,5-tris(acetyloxy)-6-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}oxan-2-yl]methyl acetate

EC Number:

930-010-9

Cas Number:

461432-25-7

Molecular formula:

C29H33ClO10

IUPAC Name:

[(2R,3R,4R,5S,6S)-3,4,5-tris(acetyloxy)-6-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}oxan-2-yl]methyl acetate

Test material form:

solid: particulate/powder

Details on test material:

Appearance: White crystalline solid
Storage conditions: At room temperature protected from light

Specific details on test material used for the study:

Test substance handling: Amber glassware was used or container was wrapped in aluminum-foil

Test animals

Species:

rat

Strain:

other: Crl:WI(Han).

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: Female animals: Charles River Deutschland, Sulzfeld, Germany. Male animals: Charles Ri ver Laboratories USA, Kingston.
- Age at study initiation:
Males: approximately 10-12 weeks.
Females: approximately 10-12 and 12-14 weeks for the pretest and main study, respectively.
- Weight at study initiation: 268-301 gr (males) or 201-231 gr (females)
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females w ere individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

ENVIRONMENTAL CONDITIONS (set onditions)
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 Februari 2016 to 28 June 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036

Details on exposure:

Method of formulation: Formulations (w/w) were prepared daily and used within 5 hours after preparat ion. The formulations were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.

Storage conditions of formulations: At room temperature protected from light.

Justification for use and choice of vehicle: Based on trial formulations performed at Charles River Den Bosch.

Dose volume: 4 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Details on mating procedure:

- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- 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 had occurred, the males and females were separated.
- After 14 days of unsuccessful pairing the female who had not shown evidence of mating was separated from her male.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples of the control group and 40 mg/kg bw/day treatment group were taken in duplicate from the middle position of the container and immediately stored on dry ice. Samples of the 4 mg/kg bw/day and 400 mg/kg bw/day treatment groups were taken in duplicate from the top, middle and bottom position of the container and immediately stored on dry ice. The samples were stored at a target temperature ≤-70°C and all samples were analyzed for BMS-587172-01.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Stability of formulations in the vehicle propylene glycol for at least 5 hours at room temperature protected from light (concentration range 1-200 mg/mL) was determined as part of the analytical method development and validation study.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to termination. Females that delivered were exposed for 51-64 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were exposed for 43 days (1 female) or 53 days (1 female). Two females (400 mg/kg bw/day dose level) were not dosed on one occasion as they were littering at the time of dosing.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

Frequency of treatment:

Once daily for 7 d/w.

Details on study schedule:

- Age at mating of the mated animals in the study: Approximately 12-16 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Doses for the main study were selected based on the results from a previous single dose oral toxicity study in Sprague-Dawley rats and an oral dose range finding (DRF) study (10-17 days) in Wistar Han rats conducted with BMS-587172-01 (see results attached). In the single-dose oral toxicity study, no clinical signs of toxicity or macroscopic abnormalities were observed at the limit dose of 2000 mg/kg. In the dose range finding study, 17 daily oral doses of 200 and 400 mg/kg/day (n=3/female/group) were well tolerated and not associated with clear clinical evidence of toxicity. Because of a lack of toxicity observed at these doses, an additional dose of 1000 mg/kg/day was added for 10 daily doses. At 1000 mg/kg/day, all animals exhibited clinical signs which included hunched posture, piloerection, increased salivation and rales. In addition, toxicity studies conducted with structurally similar compounds, BMS-587319-03 and BMS-512148-01, resulted in clinical toxicity at doses ≥ 200 mg/kg/day. Based on these data, the high dose of 400 mg/kg/day of BMS-587172-01 was selected for this study because it was anticipated to induce some evidence of toxicity, but within acceptable limits. The low dose of 4 mg/kg/day was selected because it was anticipated to be a no effect level, and the intermediate dose of 40 mg/kg/day was selected because it allowed for a 10 fold spacing between dose levels.

Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.

Identification of pups:
On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.

Selection of animals for selected measurements:
5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.

On Day 2 of the mating period, a few females which had their mating confirmed were accidentally dosed based on their body weights from the previous day instead of their most recent body weights (Day 0 post-coitum). Due to this error, 5 females across the test item-treated groups received a slightly higher dose. This deviation was not considered to have affected the study outcome as it occurred on a single day during a total treatment period of 51-64 days and the difference in applied dose volume was relative small (max. 1.25 % higher).

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily (early in the morning and close to the end of the working day).

DETAILED CLINICAL OBSERVATIONS
- Time schedule: At least once daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals, at 1 hour (± 30 minutes) after dosing (i.e. on the peak period of anticipated effects after dosing). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1, 4, 7 and 13.

FOOD CONSUMPTION
Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1, 4, 7 and 13 of lactation.

FOOD EFFICIENCY
Yes
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data.

WATER CONSUMPTION
Yes.
- Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION
No

HAEMATOLOGY
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination between 7.00 and 10.30 a.m..
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination between 7.00 and 10.30 a.m..
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked in table were: According to test guidelines

THYROID HORMONE ANALYSIS:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination between 7.00 and 10.30 a.m..
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: All adult males
- Parameters checked were: According to test guidelines

URINALYSIS
No

FUNCTIONAL OBSERVATIONS:
The following functional observation tests will be performed on each individual animal of the selected 5 animals/sex/group:
- hearing ability, pupillary reflex (L/R), static righting reflex;
- fore- and hind-limb grip strength (recorded as the mean of three measurements);
- locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system, total movements and ambulations are reported).
The selected males were tested once during Week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13).

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):

Slides of the testes were prepared for histopathological staging of spermatogenesis (5 males of the control and high dose group).

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of eight pups/litter (4/sex/litter as nearly as possible); blood samples were collected from two of the surplus pups, excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities, anogenital distance and areola/nipple retention.

- Mortality: The numbers of live and dead pups on day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on days 1, 4, 7 and 13 of lactation.
- Sex: Sex was determined for all pups on days 1 and 4 of lactation.
- 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.

THYROID HORMONE ANALYSIS:
- PND13-15 pups
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination between 7.00 and 10.30 a.m..
- How many animals: From 2 pups per litter (if possible from one male and one female)
- Parameters checked were: According to test guidelines

Postmortem examinations (parental animals):

GROSS PATHOLOGY
All animals were fasted overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.

The number of former implantation sites and corpora lutea were recorded for all paired females.

- Selected 5 animals/sex/group and all animals that died spontaneously or were killed in extremis: According to test guidelines

- All remaining animals and females which failed to deliver: According to test guidelines

ORGAN WEIGHTS
- Selected 5 animals/sex/group: According to test guidelines

- All remaining males:
Cowper’s glands, epididymides, glans penis, levator ani plus bulbocavernosus muscle complex (LABC), Testes and thyroid.

HISTOPATHOLOGY
According to test guidelines

Inadvertently, a few tissues were not available for histopathology. Reasons for missing a few tissues included that these tissues were not discernable at necropsy or trimming, or were erroneously not collected at necropsy. Missing tissues are listed in raw data and pathology report.
Evaluation: Sufficient data was available for adequate histopathological evaluation.

Postmortem examinations (offspring):

SACRIFICE
Pups, younger than 7 days were killed by decapitation.
On PND 4 (at culling), from 2 pups per litter blood samples (0.4 mL in total) were collected by decapitation between 7.00 and 10.30 a.m.. Blood samples from the 2 pups per litter were collected into one serum tube.
On PND 13-15, from 2 pups per litter (if possible from one male and one female) blood samples were collected at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane, between 7.00 and 10.30 a.m., followed by exsanguination.
All remaining pups (PND 7-15) were sacrificed.

GROSS NECROPSY
All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. At terminal sacrifice (PND 13-15), the thyroid from 1 male and 1 female pup per litter was preserved in 10% buffered formalin.

HISTOPATHOLOGY / ORGAN WEIGTHS
No.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 1; 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 (Ref. 2; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 3) was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test (Ref. 4) was applied to motor activity data to determine intergroup differences.

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 paired) x 100
- Conception 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:

- Percentage live males at First Litter Check: (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check: (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
- 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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental findings that were noted included rales, scabbing, wounds and alopecia. 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. As they were observed in the absence of a treatment related distribution and/or were seen for individual animals only, they were not considered to be test item related.

Mortality:

no mortality observed

Description (incidence):

One high dose male was euthanized on Day 9 of the pre-mating period due to problems with his hindleg, most likely caused by an accident. Clinical signs of this male included abnormal posture, abnormal gait (at a slight degree), and swelling of his left hindleg (at a slight to moderate degree) from pre-mating Day 5 onwards. The slight body weight loss (1%) recorded from premating Days 1 to 8 was probably related to a reduced food intake caused by his impaired mobility. Necropsy findings
included thickened left hindleg and isolated reddish foci on the stomach mucosa.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was a trend towards higher food consumption (absolute) of both males and females in all test item treated groups when compared to control animals. However, no clear dose relation could be established. Changes reached statistical significance for females in all dose levels during almost the entire post-coitum period, and for 400 mg/kg bw/day females during lactation days 4-7.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

There was a trend towards higher food consumption relative to body weight for both males and females in all test item treated groups when compared to control animals. However, no clear dose relation could be established. Changes reached statistical significance for females in all dose levels during almost the entire post-coitum period.

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistical significant higher urea levels were recorded for males at 40 and 400 mg/kg/day (12.2 and 12.3 mmol/L, respectively) and females at 400 mg/kg/day (15.1 mmol/L) when compared to their concurrent controls. In males (40 and 400 mg/kg bw/day), values were above the available historical range, but no dose related trend could be established. In females (400 mg/kg bw/day), mean value was at the higher end of the historical range. In the absence of any correlating changes on organ level, these changes were not considered as adverse.
Lower glucose concentration was noted in all test item treated groups, reaching statistical significance for males, but not females. No dose dependency could be established.
Any remaining statistically significant changes were considered to be chance findings as no dose related trend could be established and/or values remained within the range considered normal for rats of this age and strain. Furthermore, no correlating changes on organ level were noted.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There was a higher incidence in the follicular cell hypertrophy in the thyroid glands of males treated at 4 and 400 mg/kg/day. Based on the minimal degree, which is a common background finding in male rats, and the absence of a dose-response effect it was considered not to be related to the test item.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Serum levels of T4 in P0-males were considered not to be affected by treatment.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Most females had regular cycles of 4 days. Extended di-estrus (i.e. at least 5 consecutive days of di-estrus) during pairing occurred in one control female and two females at 40 mg/kg bw/day. The latter female with extended di-estrus had also an irregular estrous cycle (i.e. one cycle of 3 days). In addition, one female at 400 mg/kg bw/day had in irregular estrous cycle with 2 cycles of 2 days. In the absence of a dose related response, these abnormalities in estrous cycle were considered to be incidental and not related to treatment

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

All females of the control and treated groups showed evidence of mating. Mating index was 100% for all groups.
Fertility and conception index were not affected by treatment. All paired and mated females were pregnant, except for one female at 4 mg/kg bw/day. Mating was overlooked for one control female which had 1 implantation site only. The mating date of this animal was confirmed by the results of vaginal smears.
Precoital time was considered not to be affected by treatment. All females were mated within 4 days of pairing, except for one control female and two females at 40 mg/kg bw/day for which mating could not be confirmed before 12-13 days of pairing. These three females were noted with extended di-estrus during pairing. In the absence of a dose related response, no toxicological relevance was attached to this finding.
Gestation index and duration of gestation were unaffected by treatment up to 400 mg/kg bw/day. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of premature birth. No deficiencies in maternal care were observed.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicity was observed up to and including the highest dose level tested.

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental clinical symptoms of pups consisted of blue discoloration of the back, blue spots on snout, neck, back, abdomen or hindleg, scab or wound on the tip of the tail, back, abdomen or front leg, missing tail tip, autolysis, lean appearance, alopecia. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The live birth index, viability index and lactation index were considered not to be affected by treatment.
Two control pups and one and three pups in respectively the 40 and 400 mg/kg bw/day groups were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
In the 40 mg/kg bw/day group, three pups within one litter were found dead on lactation days 2 ,4 and 13, respectively. No other pups were found dead/missing in the period between first litter check on day 1 and lactation day 13.

Body weight and weight changes:

no effects observed

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 pups on PND 13-15 were considered not to be affected by treatment up to and including 400 mg/kg bw/day. The mean value for the 40 mg/kg bw/day group was statistically significantly higher when compared to the concurrent control group but this increase occurred without a dose related trend.

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Sex ratio and anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment up to 400 mg/kg bw/day. The median anogenital distance of male pups at 4 mg/kg/day was statistically significantly lower when compared to the concurrent control group but this decrease occurred without a dose related trend.
Treatment up to and including 400 mg/kg bw/day had no effect on areola/nipple retention. Nipples were not detected for any of the examined male pups on PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Incidental macroscopic findings of pups included autolysis, lean appearance, alopecia and missing tail tip. The nature and incidence of these findings remained within the range considered normal for pups of this age. They were therefore considered to be of no toxicological relevance.

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicity was observed up to and including the highest dose level tested.

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Analysis of dose preparations:

In the control group formulation, no test substance was detected.

The mean accuracies for the 4, 40 and 400 mg/kg bw/day dose levels were 99.5%, 96.2% and 97.2%, respectively. The coefficient of variantion for the formulations of the 4 and 400 mg/kg bw/day dose levels were 4.0% and 0.9%, respectively.

Applicant's summary and conclusion

Conclusions:

In an oral OECD 422 screening study, the NOAEL parental and NOAEL for reproduction were derived to be at least 400 mg/kg bw/day based on the absence of effects up to and including the highest dose level tested.