Reaction products of Dihydro-3-(tetrapropenyl)furan-2,5 dione with propane-1,2,diol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Under the conditions of this study it is concluded that the NOAEL of the test material is greater than or equal to 300 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 June 2016 to 27 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

RccHan™:WIST rat

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The RccHan™;WIST was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Males: 69 to 75 days old, Females: 62 to 68 days old
- Weight at study initiation: Males: 276 to 310 g and Females: 150 to 189 g
- Housing: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatisation, pre-pairing, gestation, littering and lactation periods. Solid bottom cages contained softwood based bark-free fibre bedding, which was changed at appropriate intervals. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing. Chew blocks and plastic shelters were provided as enrichment.
The number of animals per cage were; pre-pairing: Up to five animals of one sex; pairing: One male and one female; males after mating: Up to five animals; gestation: One female; and lactation: One female plus her litter.
- Diet: Non-restricted.
- Water: Non-restricted.
- Acclimation period: 5 days before commencement of treatment. Spare animals were removed from the study room after treatment commenced.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24 °C
- Humidity: 40 to 70 %.
- Air: Filtered fresh air which was passed to atmosphere and not recirculated, with a minimum of 15 air changes per hour.
- Photoperiod: Artificial lighting, 12 hours light: 12 hours dark.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Starting with the lowest concentration, the required amount of test material was weighed into a suitable container. Approximately 50 % of vehicle was added; it was placed in a water bath and heated up to 40 °C if necessary. It was then stirred by hand using a spatula until the formulation was at the correct consistency and then magnetically stirred until uniformly mixed. The remaining vehicle was then added to make up to the required volume and then magnetically stirred until homogenous.
- The formulation was prepared weekly.
- The formulated test material was refrigerated (nominally 2 to 8 °C).
- Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Details on mating procedure:

- M/F ratio per cage: 1:1 from within the same treatment groups.
- Length of cohabitation: Up to two weeks. Pairing commenced after a minimum of two weeks of treatment.
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the vaginal smear. Vaginal smears were taken daily after pairing until mating, using pipette lavage. Day 0 of gestation was when positive evidence of mating was detected.
- After successful mating each pregnant female was caged individually. Male/female separation occurred the day when mating evidence was detected.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

STABILITY AND HOMOGENEITY
- Before commencement of treatment, the suitability of the proposed mixing procedures was determined. In this study, homogeneity and stability of the test material in the vehicle at concentrations of 2 mg/mL and 200 mg/mL was achieved for seven days following ambient (nominally 21 °C) conditions and 15 days following refrigerated (nominally 2 to 8 °C) conditions.

ACHIEVED CONCENTRATION
- Samples of each formulation prepared for administration the first and last formulation occasions were analysed for achieved concentration of the test material.

ANALYTICAL PROCEDURE
- The analytical method involved extraction and dilution in Ethanol followed by liquid chromatographic analysis with mass spectrometric detection (LC-MS/MS). Sample Concentrations were determined with reference to single bracketing standards. Procedural recovery samples were prepared concurrently with samples as a quality control measure. Samples and standards were matrix-matched by including appropriate amounts of the initial control vehicle extract as necessary.

CONCENTRATION OF DOSE FORMULATIONS
- On the First and Last Formulation preparation occasion, samples of the formulations were collected. Formulations (4 × 1 mL, accurately weighed) were sampled from the middle of the formulation by Pharmacy personnel. Duplicate samples were analysed in accordance with the analytical procedure. The remaining samples were retained for contingency. For the First Occasion, contingency samples for Group 1 (single sample) and Group 2 and Group 3 (duplicate samples) were analysed as the original results showed mean concentrations > + 10 % from the nominal concentration. The Group 1 sample was analysed to confirm the integrity of the contingency samples by confirming a clear control sample. The remainder were disposed of once satisfactory results were achieved.

RESULTS AND CONCLUSION
- The mean concentrations were within -15 to -5 % of the nominal concentration, confirming the accuracy of formulation. Difference from the mean of individual samples were < ± 5 % (coefficient of variation was < 5 % for Groups 2 and 3 on the First Occasion) confirming precise analysis. Procedural recoveries analysed concurrently with samples were within acceptance criteria of 90 to 110 % except for the 20 mg/mL procedural recoveries in each analysis.

Duration of treatment / exposure:

- Males were treated daily for 15 days before pairing, during the pairing period, and then up to necropsy after a minimum of four consecutive weeks.
- Females were treated daily for 15 days before pairing, throughout pairing and gestation, and until Day 6 of lactation.
- The F1 generation received no direct administration of the test item. Any exposure to the test item or metabolites was through the mother to the offspring in utero and/or through the milk.

Frequency of treatment:

Once daily at approximately the same time each day.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

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
- Dose levels were selected based on the results of the two-week preliminary oral toxicity study and four-week oral toxicity study.
- In the 14-day repeat-dose dose range finding study, doses of 500 and 1 000 mg/kg/day were not tolerated. Macroscopic findings at 300 mg/kg/day comprised irregular surface of the stomach for two females and depressions and thickened stomach wall for one male. There were no treatment-related macroscopic findings for animals which received 100 mg/kg/day.
- In the 4-week repeat dose toxicity study, doses of 30, 100 and 300 mg/kg/day were well tolerated and did not result in any mortality or signs of other adverse systemic toxicity based on the study investigations performed up to the end of the 4 week treatment period and excluding results of the histopathological assessment. These findings did not preclude the use of 300 mg/kg/day as the high dose on this current Reproductive/Developmental Toxicity Screening Study.
- Based on this information, it was considered appropriate to investigate a high dose level of 300 mg/kg/day. The intermediate and low dose levels, 100 and 30 mg/kg/day, respectively, were selected to assess any dose-responsiveness of any test material-related finding.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
Clinical observations are presented for each animal that showed signs, providing detail of the type of sign, day of occurrence and information on the duration of the sign applicable.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during the first week of treatment, weekly thereafter and for females on Days 0, 7, 14 and 20 after mating and Days 1 and 6 of lactation, detailed observations were recorded at the following times in relation to dose administration: pre-dose, after completion of dosing each group, one to two hours after dosing and as late as possible in the working day.
- A detailed physical examination was performed weekly for males and for females weekly before pairing, on Days 0, 7, 14 and 20 after mating and Days 1 and 7 of lactation to monitor general health.

BODY WEIGHT: Yes
- F0 males were weighed: During acclimatisation, before dosing on the day that treatment commenced (Day 1) and weekly thereafter and on the day of necropsy.
- F0 females were weighed: During acclimatisation, before dosing on the day that treatment commenced (Day 1) and weekly before pairing, on days 0, 3, 7, 10, 14, 17 and 20 after mating, on days 1, 4, and 7 of lactation and on the day of necropsy.
Group mean values and SD were calculated from individual body weight data on each recorded occasion. For the offspring, litter mean body weight (and SD) was calculated separately for males and females; the group mean values were derived from the individual litter values.
Group mean weight changes were calculated from the weight changes of individual animals. Offspring body weight change was calculated relative to Day 1 of age.
Body weights were plotted graphically with respect to the start of dosing or the start of the relevant period.
One female (number 67 receiving 100 mg/kg/day) had only one uterine implantation, which had resorbed. This female was therefore classified as “total litter resorption” and excluded from group mean gestation body weight data as this was not a typical pregnancy.

FOOD CONSUMPTION:
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded to measure food consumption.
- For F0 males food consumption was measured weekly before pairing (from first day of treatment until pairing).
- For F0 females food consumption was measured weekly before pairing (from first day of treatment until pairing), on days 0-2, 3-6, 7-9, 10-13, 14-16 and 17-19 after mating and on days 1-3 and 4-6 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.
Group mean food consumption and standard deviation values were derived from unrounded cage values. Female 67, classified as total litter resorption, was excluded from group mean calculations of gestation food consumption data as this was not a typical pregnancy.

WATER CONSUMPTION: No

OTHER: PARTURITION AND GESTATION LENGTH
- Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
- Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

OTHER: PRE-COITAL INTERVAL
Individual intervals were tabulated for females only, for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals was calculated for durations of 1-4, 5-8, 9-12 and 13-14 days of pairing.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
- Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1 to 7 of age.
- Sex ratio of each litter: Recorded on Days 1, 4 and 7 of age.
- Individual offspring body weights: Days 1, 4 and 7 of age.

GROSS EXAMINATION OF DEAD PUPS:
- Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed for premature offspring deaths. Abnormal tissues were retained in an appropriate fixative.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals during Week 5 of respective treatment.
- Maternal animals: All surviving animals on Day 7 of lactation
- F0 females failing to produce a viable litter: Day 25 after the mating period.
All adult animals were killed by carbon dioxide asphyxiation.

GROSS NECROPSY
- All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
- Abnormalities, epididymides (caput, corpus and cauda), ovaries, pituitary, prostate, seminal vesicles (with coagulation gland), testes, uterus with cervix and oviducts and vagina were all fixed.
- For the females in each ovary the number of corpora lutea and uterine implantation sites were recorded. The number of uterine implantation sites were checked after staining with ammonium sulphide, for females failing to produce a viable litter only.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Weights of the following were recorded: epididymides (caput, corpus and cauda), ovaries, prostate, seminal vesicles (with coagulation gland) and testes. For bilateral organs, left and right organs were weighed together, unless specified. Requisite organs were weighed for animals killed at scheduled intervals. Organ weights were presented both as absolute/unadjusted and adjusted for terminal body weight, using the weight recorded on the day of necropsy.
- Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of testes which were fixed initially in modified Davidson's fluid.
- Histology and light microscopy were performed on: Abnormalities, epididymides (caput, corpus and cauda), ovaries and testes.
- Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.
- Light microscopy findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
- For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
- For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 7 days of age.
- These animals were subjected to post-mortem examinations (macroscopic and/or microscopic examination) as follows:
- The offspring were examined externally, if found to be normal offspring were discarded without further examination. Any externally abnormal offspring were also examined internally. Abnormal tissues were retained in an appropriate fixative.
- Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed. Abnormal tissues were retained in an appropriate fixative.

Statistics:

See below.

Reproductive indices:

Group values were calculated for males and females separately for the following:
- Percentage mating (%) = (Number of animals mating/ Animals paired) x 100

- Conception rate (%) = (Number of animals achieving pregnancy/ Animals mated) x 100

- Fertility index (%) = (Number of animals achieving pregnancy/ Animals pairing) x 100

Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:

- Gestation index (%) = (Number of live litters born/ Number pregnant) x 100

Offspring viability indices:

Survival Indices
The following indices were calculated for each litter:

- 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% where 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 7/ Number live offspring on Day 1 after littering) x 100

Group mean values were calculated from individual litter values.

Sex Ratio
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 and 7 of age.

Percentage males = (Number of males in litter / total number of offspring in litter) x 100

Group mean values were calculated from individual litter values.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no treatment related changes observed in association with the time of dosing.
- Salivation was observed in males and females receiving 300 mg/kg/day, and associated chin rubbing was observed in a single male at the same dose, and for several females at this dose level during gestation and lactation and to a lesser extent among females at 100 mg/kg/day. These signs are most likely to reflect a general distaste of the formulation, and are not considered a toxic effect of treatment. Piloerection was observed in two females receiving 300 mg/kg/day on Day 1 of lactation, however in light of the very low incidence on a single occasion; no effect of treatment is inferred.
- Clinical signs observed were minor and are not attributed to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- Bodyweight gain for males throughout their treatment period, and for females before pairing, and throughout gestation and lactation, was unaffected by treatment at all dose levels investigated.
- Mean values of body weight for females receiving 300 mg/kg/day were higher than Controls during Days 0 to14 of gestation, attaining statistical significance.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- There was no adverse effect of treatment upon food consumption before pairing in males or females, or during gestation and lactation for females, at any dose level.
- Mean values of food consumption for females receiving 100 or 300 mg/kg/day were slightly higher than Controls during Days 0 to 14 of gestation, attaining statistical significance on Days 7-13 of gestation.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

- There were no microscopic findings in adult males or females at necropsy that would infer any test material treatment related changes.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

- Mean pre-coital interval was unaffected by treatment. Only one pairing on study (at 300 mg/kg/day) did not mate within the first 4 days of pairing.
- Test material treatment had no effect upon mating performance or fertility and there was no effect of treatment upon mean gestation length or the gestation index.
- One female (receiving 100 mg/kg/day) failed to litter. This animal had only one uterine implantation, which had resorbed. This female was therefore classified as “total litter resorption” and excluded from group mean gestation body weight and food consumption data as this was not a typical pregnancy and it is not uncommon for animals with a very low number of implantations to show total litter resorption. All other females reared a live litter to Day 7 of lactation.
- There was no effect of test material treatment upon post-implantation survival, live birth or viability indices. Mean litter size was similar to Controls across all dose levels on Day 1 of lactation, and subsequent litter size to Day 7 of lactation was unaffected by treatment. There was no effect of treatment upon mean sex ratio at any of the dose levels investigated.
- The mean corpora lutea count was unaffected by treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Clinical signs:

no effects observed

Description (incidence and severity):

- There was no change in the clinical condition of offspring that was considered related to maternal treatment at any of the dose levels investigated.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- Mean body weight of offspring on Day 1 of age was slightly low at 300 mg/kg/day and marginally low at 100 mg/kg/day. Statistical significance was attained for male offspring in the 300 mg/kg/day group, and for female offspring in the 100 and 300 mg/kg/day groups. Subsequent body weight gain during Days 1-7 of age was similar to Controls across all treatment groups and considered unaffected by treatment at all dose levels investigated.

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):

- Necropsy findings observed in offspring dying prematurely or in those killed at scheduled termination revealed no findings that were considered to relate to parental test material treatment.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

(F1 generation were not dosed directly)

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Reproductive effects observed:

no

Conclusions:

Under the conditions of this study It is concluded that the NOAEL of the test material is greater than or equal to 300 mg/kg bw/day.

Executive summary:

The reproductive toxicity of the test material was determined in accordance with the standardised guideline OECD 421 using a reproductive/ developmental toxicity screening study in rats, under GLP conditions.

Three groups of ten male and ten female rats received the test material at doses of 30, 100 or 300 mg/kg/day by oral (gavage) administration at a dose volume of 5 mL/kg. Males were treated daily for 15 days before pairing, during the pairing period, and then up to necropsy after a minimum of four consecutive weeks. Females were treated daily for 15 days before pairing, throughout pairing and gestation, and until Day 6 of lactation. Females were allowed to litter and rear their offspring before they were killed on Day 7 of lactation. The F1 generation received no direct administration of the test material; any exposure was in utero or via the milk.  A similarly constituted Control group received the vehicle, corn oil, at the same dose volume as the treated groups.

During the study, clinical condition, body weight, food consumption, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations of organs of the reproductive system were undertaken. The clinical condition, litter size and survival, sex ratio, body weight and macropathology for all offspring were also assessed. 

The oral administration of the test material was well tolerated with no adverse effect of treatment apparent.

There was no effect of treatment upon body weight or food consumption of the adult males or females at any of the dose levels investigated. Treatment up to 300 mg/kg/day also had no effect upon mating performance, fertility, gestation length or gestation index.

Mean body weights of male and female offspring on Day 1 of age at 300 or 100 mg/kg/day were slightly/marginally lower than in Controls. However, the number of offspring born, their subsequent survival, growth and clinical condition to Day 7 of age were unaffected by treatment at dose levels up to 300 mg/kg/day. There were no macroscopic findings in offspring that were considered to relate to parental test material treatment at any of the dose levels investigated.

It is concluded that oral administration of the test material to male and female Han Wistar (RccHan™;WIST) rats for at least 4 weeks at dose levels of 30, 100 or 300 mg/kg/day was well tolerated, with no adverse effect of treatment upon reproductive or developmental parameters observed up to the highest dose level investigated of 300 mg/kg/day.

Under the conditions of this study It is concluded that the NOAEL of the test material is greater than or equal to 300 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

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 guideline OECD 421 using a reproductive/ developmental toxicity screening study in rats, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Three groups of ten male and ten female rats received the test material at doses of 30, 100 or 300 mg/kg/day by oral (gavage) administration at a dose volume of 5 mL/kg. Males were treated daily for 15 days before pairing, during the pairing period, and then up to necropsy after a minimum of four consecutive weeks. Females were treated daily for 15 days before pairing, throughout pairing and gestation, and until Day 6 of lactation. Females were allowed to litter and rear their offspring before they were killed on Day 7 of lactation. The F1 generation received no direct administration of the test material; any exposure was in utero or via the milk.  A similarly constituted Control group received the vehicle, corn oil, at the same dose volume as the treated groups.

During the study, clinical condition, body weight, food consumption, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations of organs of the reproductive system were undertaken. The clinical condition, litter size and survival, sex ratio, body weight and macropathology for all offspring were also assessed. 

There was no effect of treatment upon body weight or food consumption of the adult males or females at any of the dose levels investigated. Treatment up to 300 mg/kg/day also had no effect upon mating performance, fertility, gestation length or gestation index.

Mean body weights of male and female offspring on Day 1 of age at 300 or 100 mg/kg/day were slightly/marginally lower than in Controls. However, the number of offspring born, their subsequent survival, growth and clinical condition to Day 7 of age were unaffected by treatment at dose levels up to 300 mg/kg/day. There were no macroscopic findings in offspring that were considered to relate to parental test material treatment at any of the dose levels investigated.

It is concluded that oral administration of the test material to male and female Han Wistar (RccHan™;WIST) rats for at least 4 weeks at dose levels of 30, 100 or 300 mg/kg/day was well tolerated, with no adverse effect of treatment upon reproductive or developmental parameters observed up to the highest dose level investigated of 300 mg/kg/day.

Under the conditions of this study It is concluded that the NOAEL of the test material is greater than or equal to 300 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

Under the conditions of this study it is concluded that the NOAEL for reproductive and offspring development of the test material is greater than or equal to 300 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 June 2016 to 27 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421; Reproduction/Developmental Toxicity Screening Test

Version / remarks:

1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Males: 69 to 75 days old, Females: 62 to 68 days old.
- Weight at study initiation: Males: 276 to 310 g and Females: 150 to 189 g.
- Housing: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatisation, pre-pairing, gestation, littering and lactation periods. Solid bottom cages contained softwood based bark-free fibre bedding, which was changed at appropriate intervals. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing. Chew blocks and plastic shelters were provided as enrichment. The number of animals per cage were; pre-pairing: Up to five animals of one sex; pairing: One male and one female; males after mating: Up to five animals; gestation: One female; and lactation: One female plus her litter.
- Diet: Non-restricted.
- Water: Non-restricted.
- Acclimation period: 5 days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24 °C
- Humidity: 40 to 70 %.
- Air: Filtered fresh air which was passed to atmosphere and not recirculated, with a minimum of 15 air changes per hour.
- Photoperiod: Artificial lighting, 12 hours light: 12 hours dark.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Starting with the lowest concentration, the required amount of test material was weighed into a suitable container. Approximately 50 % of vehicle was added; it was placed in a water bath and heated up to 40 °C if necessary. It was then stirred by hand using a spatula until the formulation was at the correct consistency and then magnetically stirred until uniformly mixed. The remaining vehicle was then added to make up to the required volume and then magnetically stirred until homogenous.
- The formulation was prepared weekly.
- The formulated test material was refrigerated (nominally 2 to 8 °C).
- Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

STABILITY AND HOMOGENEITY
- Before commencement of treatment, the suitability of the proposed mixing procedures was determined. In this study, homogeneity and stability of the test material in the vehicle at concentrations of 2 mg/mL and 200 mg/mL was achieved for seven days following ambient (nominally 21 °C) conditions and 15 days following refrigerated (nominally 2 to 8 °C) conditions.

ACHIEVED CONCENTRATION
- Samples of each formulation prepared for administration the first and last formulation occasions were analysed for achieved concentration of the test material.

ANALYTICAL PROCEDURE
- The analytical method involved extraction and dilution in Ethanol followed by liquid chromatographic analysis with mass spectrometric detection (LC-MS/MS). Sample Concentrations were determined with reference to single bracketing standards. Procedural recovery samples were prepared concurrently with samples as a quality control measure. Samples and standards were matrix-matched by including appropriate amounts of the initial control vehicle extract as necessary.

CONCENTRATION OF DOSE FORMULATIONS
- On the First and Last Formulation preparation occasion, samples of the formulations were collected. Formulations (4 × 1 mL, accurately weighed) were sampled from the middle of the formulation by Pharmacy personnel. Duplicate samples were analysed in accordance with the analytical procedure. The remaining samples were retained for contingency. For the First Occasion, contingency samples for Group 1 (single sample) and Group 2 and Group 3 (duplicate samples) were analysed as the original results showed mean concentrations > + 10 % from the nominal concentration. The Group 1 sample was analysed to confirm the integrity of the contingency samples by confirming a clear control sample. The remainder were disposed of once satisfactory results were achieved.

RESULTS AND CONCLUSION
- The mean concentrations were within -15 to -5 % of the nominal concentration, confirming the accuracy of formulation. Difference from the mean of individual samples were < ± 5 % (coefficient of variation was < 5 % for Groups 2 and 3 on the First Occasion) confirming precise analysis. Procedural recoveries analysed concurrently with samples were within acceptance criteria of 90 to 110 % except for the 20 mg/mL procedural recoveries in each analysis.

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1:1 from within the same treatment groups.
- Length of cohabitation: Up to two weeks. Pairing commenced after a minimum of two weeks of treatment.
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the vaginal smear. Vaginal smears were taken daily after pairing until mating, using pipette lavage. Day 0 of gestation was when positive evidence of mating was detected.
- After successful mating each pregnant female was caged individually. Male/female separation occurred the day when mating evidence was detected.

Duration of treatment / exposure:

- Males were treated daily for 15 days before pairing, during the pairing period, and then up to necropsy after a minimum of four consecutive weeks.
- Females were treated daily for 15 days before pairing, throughout pairing and gestation, and until Day 6 of lactation.
- The F1 generation received no direct administration of the test item. Any exposure to the test item or metabolites was through the mother to the offspring in utero and/or through milk.

Frequency of treatment:

Once daily at approximately the same time each day.

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 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
- Dose levels were selected based on the results of the two-week preliminary oral toxicity study and four-week oral toxicity study.
- In the 14-day repeat-dose dose range finding study, doses of 500 and 1 000 mg/kg/day were not tolerated. Macroscopic findings at 300 mg/kg/day comprised irregular surface of the stomach for two females and depressions and thickened stomach wall for one male. There were no treatment-related macroscopic findings for animals which received 100 mg/kg/day.
- In the 4-week repeat dose toxicity study, doses of 30, 100 and 300 mg/kg/day were well tolerated and did not result in any mortality or signs of other adverse systemic toxicity based on the study investigations performed up to the end of the 4 week treatment period and excluding results of the histopathological assessment. These findings did not preclude the use of 300 mg/kg/day as the high dose on this current Reproductive/Developmental Toxicity Screening Study.
- Based on this information, it was considered appropriate to investigate a high dose level of 300 mg/kg/day. The intermediate and low dose levels, 100 and 30 mg/kg/day, respectively, were selected to assess any dose-responsiveness of any test material-related finding.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s).
Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
Clinical observations are presented for each animal that showed signs, providing detail of type of sign, day of occurrence and information on the duration of the sign applicable.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during the first week of treatment, weekly thereafter and for females on Days 0, 7, 14 and 20 after mating and Days 1 and 6 of lactation, detailed observations were recorded at the following times in relation to dose administration: Pre-dose, after completion of dosing each group, one to two hours after dosing and as late as possible in the working day.
- A detailed physical examination was performed weekly for females weekly before pairing, on Days 0, 7, 14 and 20 after mating and Days 1 and 7 of lactation to monitor general health.

BODY WEIGHT: Yes
- F0 females were weighed: During acclimatisation, before dosing on the day that treatment commenced (Day 1) and weekly before pairing, on days 0, 3, 7, 10, 14, 17 and 20 after mating, on days 1, 4, and 7 of lactation and on the day of necropsy.
Group mean values and SD were calculated from individual body weight data on each recorded occasion. For the offspring, litter mean body weight (and SD) was calculated separately; the group mean values were derived from the individual litter values.
Group mean weight changes were calculated from the weight changes of individual animals. Offspring body weight change was calculated relative to Day 1 of age.
Body weights were plotted graphically with respect to the start of dosing or the start of the relevant period.
One female (number 67 receiving 100 mg/kg/day) had only one uterine implantation, which had resorbed. This female was therefore classified as “total litter resorption” and excluded from group mean gestation body weight data as this was not a typical pregnancy.

FOOD CONSUMPTION:
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded to measure food consumption.
- The F0 females food consumption was measured Weekly before pairing (from first day of treatment until pairing), on days 0-2, 3-6, 7-9, 10-13, 14-16 and 17-19 after mating and on days 1-3 and 4-6 of lactation.
Group mean food consumption and standard deviation values were derived from unrounded cage values. Female 67, classified as total litter resorption, was excluded from group mean calculations of gestation food consumption data as this was not a typical pregnancy.
From these records, the mean weekly or daily consumption per animal (g/animal/week) or g/animal/day) was calculated for each phase.

WATER CONSUMPTION: No

PARTURITION AND GESTATION LENGTH
- Time elapsing between the detection of mating and commencement of parturition.
- From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on: Maternal animals: All surviving animals on Day 7 of lactation and F0 females failing to produce a viable litter: Day 25 after the mating period.
- Organs examined: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
- Abnormalities, ovaries, pituitary, uterus with cervix and oviducts and vagina were all fixed.
- The number of corpora lutea and uterine implantation sites were recorded for each ovary. The number of uterine implantation sites were checked after staining with ammonium sulphide, for females failing to produce a viable litter only.
- Weights of the ovaries were recorded.
- Histology and light microscopy were performed on: Abnormalities and ovaries
- Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required. Sections were stained with haematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.
- Light microscopy findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
- For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.

OTHER: PRE-COITAL INTERVAL
Individual intervals were tabulated for females only, for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals was calculated for durations of 1-4, 5-8, 9-12 and 13-14 days of pairing.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Weight of ovaries.
- The number of corpora lutea and uterine implantation sites were recorded.
- The number of uterine implantation sites were checked after staining with ammonium sulphide, for females failing to produce a viable litter only.

Fetal examinations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
- Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1 to 7 of age.
- Sex ratio of each litter: Recorded on Days 1, 4 and 7 of age.
- Individual offspring body weights: Days 1, 4 and 7 of age.

GROSS EXAMINATION OF DEAD PUPS:
- Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed for premature offspring deaths. Abnormal tissues were retained in an appropriate fixative.

SACRIFICE
- The F1 offspring were sacrificed at 7 days of age.
- These animals were subjected to post-mortem examinations (macroscopic and/or microscopic examination) as follows:
- The offspring were examined externally, if found to be normal offspring were discarded without further examination. Any externally abnormal offspring were also examined internally. Abnormal tissues were retained in an appropriate fixative.
- Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed. Abnormal tissues were retained in an appropriate fixative.

Statistics:

See below.

Indices:

Reproductive indices:
Group values were calculated for males and females separately for the following:
- Percentage mating (%) = (Number of animals mating/ Animals paired) x 100

- Conception rate (%) = (Number of animals achieving pregnancy/ Animals mated) x 100

- Fertility index (%) = (Number of animals achieving pregnancy/ Animals pairing) x 100

Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:

- Gestation index (%) = (Number of live litters born/ Number pregnant) x 100

Offspring viability indices:
Survival Indices
The following indices were calculated for each litter:

- 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% where 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 7/ Number live offspring on Day 1 after littering) x 100

Group mean values were calculated from individual litter values.

Sex Ratio
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 and 7 of age.

Percentage males = (Number of males in litter / total number of offspring in litter) x 100

Group mean values were calculated from individual litter values.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no treatment related changes observed in association with the time of dosing.
- Salivation was observed in females receiving 300 mg/kg/day, and associated chin rubbing was observed for several females at this dose level during gestation and lactation and to a lesser extent among females at 100 mg/kg/day. These signs are most likely to reflect a general distaste of the formulation, and are not considered a toxic effect of treatment. Piloerection was observed in two females receiving 300 mg/kg/day on Day 1 of lactation, however in light of the very low incidence on a single occasion; no effect of treatment is inferred.
- Clinical signs observed were minor and are not attributed to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

- There were no unscheduled deaths.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- Bodyweight gain for females before pairing, and throughout gestation and lactation, was unaffected by treatment at all dose levels investigated.
- Mean values of body weight for females receiving 300 mg/kg/day were higher than Controls during Days 0 to14 of gestation, attaining statistical significance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- There was no adverse effect of treatment upon food consumption before pairing in females, or during gestation and lactation, at any dose level.
- Mean values of food consumption for females receiving 100 or 300 mg/kg/day were slightly higher than Controls during Days 0 to 14 of gestation, attaining statistical significance on Days 7-13 of gestation.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

- There were no clear effects of test material treatment upon female ovary weights.

Gross pathological findings:

no effects observed

Description (incidence and severity):

- There was no indication of any test material treatment related changes detected in adult females at necropsy.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

- There were no microscopic findings in adult females at necropsy that would infer any test material treatment related changes.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

- There was no effect of test material treatment upon post-implantation survival, live birth or viability indices.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

- One female (receiving 100 mg/kg/day) failed to litter. This animal had only one uterine implantation, which had resorbed. This female was therefore classified as “total litter resorption” and excluded from group mean gestation body weight and food consumption data as this was not a typical pregnancy and it is not uncommon for animals with a very low number of implantations to show total litter resorption. All other females reared a live litter to Day 7 of lactation.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

- there was no effect of treatment upon mean gestation index.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

- There was no effect of treatment upon mean gestation length
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): - There was no effect of treatment upon mean gestation length

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

- Mean pre-coital interval was unaffected by treatment. Only one pairing on study (at 300 mg/kg/day) did not mate within the first 4 days of pairing.
- Test material treatment had no effect upon mating performance
- One female (receiving 100 mg/kg/day) failed to litter. This animal had only one uterine implantation, which had resorbed. This female was therefore classified as “total litter resorption” and excluded from group mean gestation body weight and food consumption data as this was not a typical pregnancy and it is not uncommon for animals with a very low number of implantations to show total litter resorption. All other females reared a live litter to Day 7 of lactation.

Other effects:

no effects observed

Description (incidence and severity):

- The mean corpora lutea count was unaffected by treatment.

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed.

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

- Mean body weight of offspring on Day 1 of age was slightly low at 300 mg/kg/day and marginally low at 100 mg/kg/day. Statistical significance was attained for male offspring in the 300 mg/kg/day group, and for female offspring in the 100 and 300 mg/kg/day groups. Subsequent body weight gain during Days 1-7 of age was similar to Controls across all treatment groups and considered unaffected by treatment at all dose levels investigated.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): - Mean body weight of offspring on Day 1 of age was slightly low at 300 mg/kg/day and marginally low at 100 mg/kg/day. Statistical significance was attained for male offspring in the 300 mg/kg/day group, and for female offspring in the 100 and 300 mg/kg/day groups. Subsequent body weight gain during Days 1-7 of age was similar to Controls across all treatment groups and considered unaffected by treatment at all dose levels investigated.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

- There was no effect of test material treatment upon the live birth or viability indices.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

-There was no effect of treatment upon mean sex ratio at any of the dose levels investigated.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

- Mean litter size was similar to Controls across all dose levels on Day 1 of lactation, and subsequent litter size to Day 7 of lactation was unaffected by treatment.

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

- Necropsy findings observed in offspring dying prematurely or in those killed at scheduled termination revealed no findings that were considered to relate to parental test material treatment.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

Under the conditions of this study it is concluded that the NOAEL for reproductive and offspring development of the test material is greater than or equal to 300 mg/kg bw/day.

Executive summary:

The developmental toxicity of the test material was determined in accordance with the standardised guideline OECD 421 using a reproductive/ developmental toxicity screening study in rats, under GLP conditions.

Three groups of ten male and ten female rats received the test material at doses of 30, 100 or 300 mg/kg/day by oral (gavage) administration at a dose volume of 5 mL/kg. Males were treated daily for 15 days before pairing, during the pairing period, and then up to necropsy after a minimum of four consecutive weeks. Females were treated daily for 15 days before pairing, throughout pairing and gestation, and until Day 6 of lactation. Females were allowed to litter and rear their offspring before they were killed on Day 7 of lactation. The F1 generation received no direct administration of the test material; any exposure was in utero or via the milk.  A similarly constituted Control group received the vehicle, corn oil, at the same dose volume as the treated groups.

During the study, clinical condition, body weight, food consumption, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations of organs of the reproductive system were undertaken. The clinical condition, litter size and survival, sex ratio, body weight and macropathology for all offspring were also assessed. 

The oral administration was well tolerated with no adverse effect of treatment apparent.

There was no effect of treatment upon body weight or food consumption of the adult females at any of the dose levels investigated. Treatment up to 300 mg/kg/day also had no effect upon mating performance, fertility, gestation length or gestation index.

Mean body weights of male and female offspring on Day 1 of age at 300 or 100 mg/kg/day were slightly/marginally lower than in Controls. However, the number of offspring born, their subsequent survival, growth and clinical condition to Day 7 of age were unaffected by treatment at dose levels up to 300 mg/kg/day. There were no macroscopic findings in offspring that were considered to relate to parental test material treatment at any of the dose levels investigated.

It is concluded that oral administration of the test material to male and female Han Wistar (RccHan™;WIST) rats for at least 4 weeks at dose levels of 30, 100 or 300 mg/kg/day was well tolerated, with no adverse effect of treatment upon reproductive or developmental parameters observed up to the highest dose level investigated of 300 mg/kg/day.

Under the conditions of this study It is concluded that the NOAEL for reproductive and offspring development of the test material is greater than or equal to 300 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

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 guideline OECD 421 using a reproductive/ developmental toxicity screening study in rats, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Three groups of ten male and ten female rats received the test material at doses of 30, 100 or 300 mg/kg/day by oral (gavage) administration at a dose volume of 5 mL/kg. Males were treated daily for 15 days before pairing, during the pairing period, and then up to necropsy after a minimum of four consecutive weeks. Females were treated daily for 15 days before pairing, throughout pairing and gestation, and until Day 6 of lactation. Females were allowed to litter and rear their offspring before they were killed on Day 7 of lactation. The F1 generation received no direct administration of the test material; any exposure was in utero or via the milk.  A similarly constituted Control group received the vehicle, corn oil, at the same dose volume as the treated groups.

During the study, clinical condition, body weight, food consumption, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations of organs of the reproductive system were undertaken. The clinical condition, litter size and survival, sex ratio, body weight and macropathology for all offspring were also assessed. 

There was no effect of treatment upon body weight or food consumption of the adult females at any of the dose levels investigated. Treatment up to 300 mg/kg/day also had no effect upon mating performance, fertility, gestation length or gestation index.

Mean body weights of male and female offspring on Day 1 of age at 300 or 100 mg/kg/day were slightly/marginally lower than in Controls. However, the number of offspring born, their subsequent survival, growth and clinical condition to Day 7 of age were unaffected by treatment at dose levels up to 300 mg/kg/day. There were no macroscopic findings in offspring that were considered to relate to parental test material treatment at any of the dose levels investigated.

It is concluded that oral administration of the test material to male and female Han Wistar (RccHan™;WIST) rats for at least 4 weeks at dose levels of 30, 100 or 300 mg/kg/day was well tolerated, with no adverse effect of treatment upon reproductive or developmental parameters observed up to the highest dose level investigated of 300 mg/kg/day.

Under the conditions of this study It is concluded that the NOAEL for reproductive and offspring development of the test material is greater than or equal to 300 mg/kg bw/day.

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 toxicity.