Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27-11-2015 to 20-07-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27-11-2015 to 20-07-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: December 2016 ; signature: March 2017
Limit test:
no
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: Not applicable.
- Specific activity: Not applicable.
- Locations of the label: Not applicable.
- Expiration date of radiochemical substance: Not applicable.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature.
- Stability under test conditions: Stable.
- Solubility and stability of the test substance in the solvent/vehicle: Formulations up to 200 mg/mL in Arachis oil BP was achieved during formulation analysis. Formulations were found to be stable in Arachis oil BP, when assessed following storage at ambient temperature (nominally 21°C) for up to two days, and refrigeration (nominally 2 - 8°C) for up to 15 days.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer.
- Preliminary purification step (if any): Not applicable.
- Final dilution of a dissolved solid, stock liquid or gel: A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item within Arachis Oil BP.
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid formulations in vehicle, of the solid test item.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): Not applicable.

OTHER SPECIFICS: Not applicable.
Species:
rat
Strain:
other: Han Wistar: RccHan: WIST
Details on species / strain selection:
The species and strain was selected in accordance with the OECD TG 421 and the other relevant guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: (P) Males 70 to 76 days (ca. 10 wks) Females 77 to 83 days (ca. 10 to 11 wks)
- Weight at study initiation: (P) Males: 270 - 314 g; Females: 172 - 216 g
- Fasting period before study: No.
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing. Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week. Cage distribution was randomised. The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups. Environmental enrichment: Aspen chew block provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary. Plastic shelter provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.
- Use of restrainers for preventing ingestion (if dermal): Not applicable.
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Males: six days before commencement of treatment. Females: 20 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: Feed: SDS VRF1 Certified pelleted diet – batch numbers and certificates of analysis provided in the full study report. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: Males: From: 2016-01-06 To: 2016-03-04 / Females: From: 2016-01-20 To: 2016-03-21
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a suspension in Arachis oil BP. The stability and homogeneity of the test item formulations were determined. Formulations up to 200 mg/mL in Arachis oil BP was achieved during formulation analysis. Formulations were found to be stable in Arachis oil BP, when assessed following storage at ambient temperature (nominally 21°C) for up to two days, and refrigeration (nominally 2 - 8°C) for up to 15 days.

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Applicant assessment indicates: Arachis oil BP was considered as appropriate based on test item solubility.
- Concentration in vehicle: Samples of each formulation prepared for administration in Week 1 of treatment, Week 4 of treatment for males and on Day 12 of lactation for females were analyzed for achieved concentration of the test item (method of analysis provided in full study report). Arachis oil formulations was assessed and confirmed at nominal concentrations of 1 mg/mL and 200 mg/mL. Test formulations analyzed for the study indicated the test item concentration was within ±10% of nominal concentrations, confirming accurate formulation. The test item concentrations for each group are indicated in table 1.
- Amount of vehicle (if gavage): Treatment volume was 4 mL/kg for control (negative, untreated group) and all treatment groups with applicable test item concentrations per group.
- Purity: BP grade Arachis Oil
- Other: Dose-formulations were analysed during the study and were reported as with ± 10% applied limits.
Details on mating procedure:
- M/F ratio per cage: 1:1 within the same treatment groups.
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: Ejected copulation plug and sperm in vaginal smear. Day 0 of gestation was when positive evidence of mating was detected (daily checks).
- Further matings after unsuccessful attempts: No.
- After successful mating each pregnant female was caged (how): housed individually in a cages comprised of a polycarbonate body with a stainless steel mesh lid.
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The homogeneity and stability was confirmed in Arachis oil BP formulations at nominal concentrations of 1 mg/mL and 200 mg/mL , during ambient temperature (nominally 21°C) for up to two days and refrigerated (nominally 2-8°C) for up to 15 days.
- The analysis consisted of GC FID analysis with internal calibration (Tetradecane in acetone ca. 2 mg/mL) within a dedicated formulation analysis report attached to the full study report. Samples of Arachis oil BP were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations along with internal standard for the calibration standards. These were then subjected to analysis by GC FID. The analytical method was validated (details available within the full study report). With LOD = 1.0167 μg/mL and LOQ = 3.389 μg/mL; linearity = > 0.999 and repeatability (n=6) of < 2%. Accuracy and precision was confirmed and mean procedural recovery was 94.1% (n=5) at 1 mg/mL and 95.6% (n=5) at 200 mg/mL.
- Mean concentrations of dose-formulations analysed during the study were within ± 10% applied limits confirming accurate test item/vehicle formulation.
Duration of treatment / exposure:
Males: Two weeks pre-pairing up to necropsy after minimum of five weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation.
Frequency of treatment:
Daily; at approximately the same time each day.
F1 generation were not dosed.
Details on study schedule:
- Age at mating of the mated animals in the study: F0 generation: Males: ca. 12 weeks ; Females: ca. 13 weeks. (i.e. after two weeks treatment).
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 - Vehicle Control Group
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Group 2 - Low Treatment Group
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3 - Intermediate Treatment Group
Dose / conc.:
650 mg/kg bw/day (nominal)
Remarks:
Group 4 - High Treatment Group
No. of animals per sex per dose:
10 per sex per dose (10 male / 10 female)
F1 generation were not dosed.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a previously conducted 28-day toxicity study (Report number attached to and cited in the full study report). The dose levels selected for the study were the same as used in the previous study. No findings in which would preclude the use of the associated dose levels.
- Rationale for animal assignment (if not random): Randomly assigned
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (and during acclimatisation period, once daily).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Signs associated with dosing: Week 1: Daily ; Week 2: Weekly; For F0 females: then Day 0, 7, 14 and 20 in Gestation Phase and Day 1, 7 and 13 in Lactation Phase. Detailed physical examinations: Weekly (F0 males), once each week (F0 females), then Day 0, 7, 14 and 20 in Gestation Phase, Day 1, 7 and 13 in Lactation Phase.

BODY WEIGHT: Yes
- Time schedule for examinations: F0 males: before dosing, and weekly thereafter. Day of necropsy ; F0 females: before dosing, and weekly until pairing, then days 0, 3, 7, 10, 14, 17 and 20 after mating, and then day 1, 4, 7, 13 and 14 of lactation plus day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not applicable.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable.
- Other: Food consumption was monitored during the study: F0 males: weekly before pairing ; F0 females: weekly before pairing, from the day that treatment commenced. Days 0-2, 3-6, 7-9, 10-13, 14-16 and 17-19 after mating. Days 1-3, 4-6 and 7-13 of lactation. Mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable.
- Time schedule for examinations: Not applicable.

OTHER:
1, See ‘FOOD CONSUMPTION’ field.
2. THYROID HORMONE ANALYSIS: Yes
- Time schedule: Day 4 of age (F1 offspring, two females per litter (where possible). Pooled sample by litter) ; Day 13 of age: Day 13 of age (F1 offspring, two males and two females per litter (where possible) ; Termination (All F0 males and F0 females)
Oestrous cyclicity (parental animals):
Dry and wet smears were taken as follows:

Dry smears:
For 15 days before pairing using cotton swabs (reproductive phase females only).

Wet smears:
Using pipette lavage during the following phases:
- For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to the Reproductive phase of the study.
- After pairing until mating (reproductive phase females only).
- For four days before scheduled termination (nominally day 11 to 14 of lactation)
Sperm parameters (parental animals):
Parameters examined in F0 male parental generations:
testis weight, epididymis weight and histology/histopathology

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.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: clinical observations, litter size, sex ratio, survival indices, ano-genital distance, body weight change, presence of nipple/areolae count in male offspring , macroscopic pathology / abnormalities. Histopathology. Thyroid Hormone Analysis.

GROSS EXAMINATION OF DEAD PUPS:
Yes, where required or if applicable.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: F0 Males: After 5 weeks treatment.
- Maternal animals: F0 females failing to produce a viable litter: Day 25 after mating. F0 females: Day 14 of lactation.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues were prepared for microscopic examination and weighed, respectively. For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed at scheduled intervals.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Particular attention was paid to the external genitalia. Thyroids were retained from one male and one female offspring in each litter on Day 13 of age.

HISTOPATHOLOGY / ORGAN WEIGHTS
The thyroid tissues were prepared for microscopic examination from one male and one female offspring in each litter. Organ weights were not performed for F1 males or F1 females.
Statistics:
All statistical analyses were carried out separately for males and females. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/foetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

The following data types were analyzed at each timepoint separately:
Body weight, using absolute weights and gains over appropriate study periods
Food consumption, over appropriate study periods during gestation and lactation
Litter size, survival indices and sex ratio
Ano-genital distance
Organ weights, both absolute and adjusted for terminal body weight

Comparisons were performed:
Group 1 vs 2, 3 and 4

Typical statistical analysis included:
parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937)
For all other comparisons the F1 approximate test was applied. Which included:
parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972)
If the F1 approximate test was significant, Dunnett's test (Dunnett 1955, 1964) was performed instead

Additional tests, included Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953), Wilcoxon rank sum tests (Wilcoxon 1945) and/or Shirley's test (Shirley 1977) and Steel's test (Steel 1959), as appropriate.

For litter size and survival indices, Fisher’s exact tests (Fisher 1973).

Sex ratio were analyzed equivalent to Lipsitz (Lipsitz 1991). Each treated group was compared to control using a Wald chi-square test.

For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied.
Reproductive indices:
Mating performance, fertility, gestation length, gestation index
Offspring viability indices:
Post-implantation survival index, viability index, lactation index and sex ratio
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 650 mg/kg bw/day dose level: chin rubbing and/or salivation for males and females and underactivity and unsteady muscle reaction were seen in some males and females between Days 1 and 6 of treatment. One male and one female showed a poor righting reflex on Day 4 of treatment. At 300 mg/kg/day chin rubbing was seen in males and females. In lactation, chin rubbing and salivation continued to affect some females. In addition, one female receiving 650 mg/kg/day (80) was observed to be underactive, struggling with dosing and lying prostrate on Day 5 or 6 of lactation.
At 300 mg/kg bw/day dose level: isolated incidences of chin rubbing for females receiving 300 mg/kg/day and three females salivating on Day 20 of gestation. Some females receiving the dose showed chin rubbing and salivation, in lactation.
One male (4M 34) receiving 650 mg/kg/day was observed to have limited use of the right hind limb on Days 4-9 of treatment and one female (2F 60) receiving 30 mg/kg/day was observed to have struggled with dosing on Day 14 after mating. These signs were considered incidental and unrelated to treatment.
There were no clinical signs at detailed physical examination associated with treatment of the test item.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths in the F0 generation.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The group mean body weights of males and females were similar across all groups at the start of treatment.
At 650 mg/kg bw/day dose level: the body weight gain of males was low when compared with that of the Controls in Week 1 but it was similar to that of the Controls in Weeks 2-5 of treatment. The body weight gain of females was slightly higher than Controls prior to pairing. No dose response was apparent, there were no consistent intergroup differences and no effect of treatment is inferred. There was no effect of treatment on body weight or body weight gains for females receiving the test item during gestation or lactation.
At 300 and 30 mg/kg bw/day dose levels: the body weight gains of males were similar to that of the Controls in Weeks 1, 3 and 4, marginally higher than that of the Control in Week 2 and increased in Week 5. No dose response was apparent and no effect of treatment is inferred. The body weight gain of females was slightly higher than Controls prior to pairing. No dose response was apparent, there were no consistent intergroup differences and no effect of treatment is inferred. There was no effect of treatment on body weight or body weight gains for females receiving the test item during gestation or lactation, with the exception of 30 mg/kg bw/day between Days 13 to 14 of lactation (after removal of the offspring) which was slightly lower than that of Controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 650 mg/kg bw/day dose level: the food consumption of males receiving the test item was lower than that of the Controls in Week 1 of treatment and marginally lower than that of Controls in Week 2. It was comparable to controls in weeks 2 to 5 of treatment. Females food consumption was also slightly lower than that of the Controls in Week 1 of treatment but similar to that of the Controls in Week 2 of treatment. Although was similar to that of Controls throughout gestation. It was slightly higher than that of control animals during lactation. This may be a result of the slightly larger litter size for females receiving the test item.

Overall, the food consumption of females receiving the test item at 30, 300 or 650 mg/kg/day was similar to that of Controls throughout gestation with the exception of females receiving 30 mg/kg/day from Days 0 - 2 where food consumption was marginally higher.
Food efficiency:
not examined
Description (incidence and severity):
See 'Food consumption'
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 treatment related changes seen in histopathology related to treatment in males terminated after 5 weeks of treatment, and females terminated on Day 14 of lactation.

See 'Reproductive Function: Sperm Measures:

At 650 mg/kg bw/day dose level: there was an incidental finding that was considered unrelated to treatment.

One control male and one male given 650 mg/kg/day had microscopic findings in the testes and epididymides. These were not considered to be related to treatment. The control male (number 4) had slight bilateral seminiferous tubular atrophy in the testes, slight reduction in spermatozoa and slight cell debris in the epididymal lumen.
The male treated with 650 mg/kg/day (number 31) had severe bilateral seminiferous tubular atrophy in the testes, severe reduction in the numbers of spermatozoa and slight cell debris in the lumen of the epididymides. These microscopic changes correlate with the macroscopic findings of small testes and small epididymides. The microscopic changes seen in the testes and epididymis of the male given 650 mg/kg/day (number 31) are not considered to be related to treatment as only one male was affected and all germ cell layers in the testes were atrophied. Extensive tubular atrophy, often involving complete loss of all germ cells from all tubules in one or both testes, can sometimes be seen as a background finding in young adult rats.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment related changes seen in histopathology related to treatment in males terminated after 5 weeks of treatment, and females terminated on Day 14 of lactation.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
THYROID HORMONE ANALYSIS
There was no clear effect of treatment for the test item at 30, 300 or 650 mg/kg/day on the thyroid hormone levels of F0 adult males at termination or F1
offspring on Day 13 of age. There was a high degree of inter-individual variability, with atypically high values seen for the following:
1. Female offspring from Litter 45 (Control) had atypically high plasma T4 on Day 13 of age (8.65 ng/mL)
2. 30 mg/kg bw/day - group 2M number 15 had atypically high plasma T4 at termination (57.8 ng/mL)
3. Control - group 1M number 3 had atypically high plasma TSH at termination (5490 pg/mL)
4. Female offspring from Litter 41 (Control) had atypically high plasma TSH on Day 13 of age (4140 pg/mL)
5. 300 mg/kg bw/day - group 3M number 30 had atypically high plasma TSH at termination (4920 pg/mL)
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
All females showed normal estrous cycles before treatment. Estrous cyclicity, pre-coital interval, fertility and mating performance were unaffected by treatment. All females were not cycling before termination (Days 11-14 of lactation), and were in dioestrous at termination.
Gestation length and gestation index were unaffected by treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no treatment related findings. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

At 650 mg/kg bw/day dose level: there was an incidental finding that was considered unrelated to treatment.

One control male and one male given 650 mg/kg/day had microscopic findings in the testes and epididymides. These were not considered to be related to treatment. The control male (number 4) had slight bilateral seminiferous tubular atrophy in the testes, slight reduction in spermatozoa and slight cell debris in the epididymal lumen.
The male treated with 650 mg/kg/day (number 31) had severe bilateral seminiferous tubular atrophy in the testes, severe reduction in the numbers of spermatozoa and slight cell debris in the lumen of the epididymides. These microscopic changes correlate with the macroscopic findings of small testes and small epididymides. The microscopic changes seen in the testes and epididymis of the male given 650 mg/kg/day (number 31) are not considered to be related to treatment as only one male was affected and all germ cell layers in the testes were atrophied. Extensive tubular atrophy, often involving complete loss of all germ cells from all tubules in one or both testes, can sometimes be seen as a background finding in young adult rats.
Reproductive performance:
no effects observed
Description (incidence and severity):
There was not affect on fertility or mating performance related to treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 650 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment related clinical signs related to treatment.
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no treatment related mortality associated with treatment within F1 males or females.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Male and female offspring body weight on Day 1 of age was similar in all groups.
Body weight gain of male and female offspring in the 650 mg/kg/day group was lower than that of the Controls, and of male and female offspring in the 30 and 300 mg/kg/day groups.

The mean absolute body weights of males and females on Day 13 of age in the 650 mg/kg bw/day group were 11% and 10% respectively lower than in Controls. However, no relationship between parental treatment at 650 mg/kg/day and the slightly lower weight gain of individual offspring is inferred since: 1. the differences in mean body weight gain during Days 1-13 of age were slight and did not attain statistical significance and 2. mean live litter size was slightly higher in the 650 mg/kg/day group compared with Controls due to chance (a marginally higher mean number of implantations and marginally higher post implantation survival of conceptuses) and this would have resulted in slightly greater within-litter competition which would be expected to be reflected in slightly lower weight gain of the individual offspring. This hypothesis is strongly supported by the fact that total growth of the litters in the 650 mg/kg/day group are similar to controls (173 g versus 167 g in controls).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment related changes seen macroscopically related to treatment in in offspring that either died prematurely or at scheduled termination on day 13. No findings were attributable to treatment.

Ano-genital distances for male or female offspring were unaffected by treatment with test item at 30, 300 or 650 mg/kg/day. A check was performed to assess for the presence or absence of nipple/areolae for the male offspring; no nipples were found.
Histopathological findings:
no effects observed
Description (incidence and severity):
See 'Other Effects' for Thyroid Hormone Analysis and related Histopathology

The thyroids of the F1 offspring killed on Day 13 of age were examined for follicular cell hypertrophy and hyperplasia and no evidence of these changes was present. Follicular lumen size/amount of colloid was semi-quantitatively graded using a five-point scale. There were no
treatment related differences in this finding.
Other effects:
no effects observed
Description (incidence and severity):
1. THYROID HORMONE ANALYSIS
There was no clear effect of treatment for the test item at 30, 300 or 650 mg/kg/day on the thyroid hormone levels of F0 adult males at termination or F1
offspring on Day 13 of age. There was a high degree of inter-individual variability, with atypically high values seen for the following:
1. Female offspring from Litter 45 (Control) had atypically high plasma T4 on Day 13 of age (8.65 ng/mL)
2. 30 mg/kg bw/day - group 2M number 15 had atypically high plasma T4 at termination (57.8 ng/mL)
3. Control - group 1M number 3 had atypically high plasma TSH at termination (5490 pg/mL)
4. Female offspring from Litter 41 (Control) had atypically high plasma TSH on Day 13 of age (4140 pg/mL)
5. 300 mg/kg bw/day - group 3M number 30 had atypically high plasma TSH at termination (4920 pg/mL)

In the related histopathology:

The thyroids of the F1 offspring killed on Day 13 of age were examined for follicular cell hypertrophy and hyperplasia and no evidence of these changes was present. Follicular lumen size/amount of colloid was semi-quantitatively graded using a five-point scale.

There were no treatment related differences in this finding.

2. Offspring Ano-Genital Distance and Male Nipple Counts
Ano-genital distances for male or female offspring were unaffected by treatment with test item at 30, 300 or 650 mg/kg/day. A check was performed to assess for the presence or absence of nipple/areolae for the male offspring; no nipples were found.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 650 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity and for reproductive / developmental toxicity for males and females is considered to be 650 mg/kg body weight per day.
Executive summary:

The study was performed according the requirements of OECD TG 421 guideline under GLP conditions. The purpose of this study was to assess general systemic toxic potential in rats, including a screening test for reproductive / development effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item by oral gavage administration for a least four weeks in Wistar Han : RccHan : WIST rats. Three groups, each comprising ten male and ten female rats, received test item at doses of 30, 300 or 650 mg/kg/day. A control group of ten males and ten females was similarly dosed with vehicle alone (Arachis oil BP), at the same dose volume as the treated groups. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were terminated on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure, if it occurred was in utero or via the milk. The F1 offspring were killed on Day 13 of age. Clinical condition, body weight change, food consumption, estrous cycles, pre-coital intervals, mating performance, fertility, gestation length, thyroid hormone analysis, organ weight, and macroscopic pathology and histopathology investigations were undertaken. For all offspring, clinical condition, litter size and survival, sex ratio, ano-genital distance, body weight and macropathology were also assessed. Nipple counts were performed on male offspring at day 13. The mean concentrations of the test item in test formulations were analyzed for the study and were within ±10% of nominal concentrations, confirming accurate formulation. The test item was determined to be stable at ambient storage for 2 days and refrigerated storage for up to 15 days within arachis oil formulations. There was no clear effect of treatment with the test item at 30, 300 or 650 mg/kg/day on the thyroid hormone levels of adult males at termination or F1 offspring on Day 13 of age. There was no effect on clinical condition or survival of males or, of females prior to pairing and during lactation, oestrus cycles, mating performance, fertility, gestation length or index, offspring survival, clinical condition, ano-genital distance or nipple counts, or macroscopic or microscopic pathology changes. Group mean body weights of males and females were similar across all groups at the start of treatment. Body weight gain of males receiving the test item at 650 mg/kg/day was low when compared with that of the Controls in Week 1 but was similar to that of the Controls in Weeks 2-5 of treatment. Transient post dosing sign included chin rubbing/salivation, underactivity and unsteady muscle reaction at 650 mg/kg/day, and chin rubbing/salivation at 300 mg/kg/day. The food consumption of males and females receiving 650 mg/kg/day was slightly low during the first week of treatment. There was considered to be no effect of treatment on the offspring sex ratio or body weight/weight gain. Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity and for reproductive / developmental toxicity was regarded to be 650 mg/kg/day for males/females.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: December 2016 ; signature: March 2017
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-methoxy-4-methylphenyl methyl carbonate
EC Number:
700-673-7
Cas Number:
132638-45-0
Molecular formula:
C10H12O4
IUPAC Name:
2-methoxy-4-methylphenyl methyl carbonate
Test material form:
solid
Details on test material:
- Physical state: Very pale yellow solid
- Storage condition of test material: Room temperature.
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: Not applicable.
- Specific activity: Not applicable.
- Locations of the label: Not applicable.
- Expiration date of radiochemical substance: Not applicable.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature.
- Stability under test conditions: Stable.
- Solubility and stability of the test substance in the solvent/vehicle: Formulations up to 200 mg/mL in Arachis oil BP was achieved during formulation analysis. Formulations were found to be stable in Arachis oil BP, when assessed following storage at ambient temperature (nominally 21°C) for up to two days, and refrigeration (nominally 2 - 8°C) for up to 15 days.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer.
- Preliminary purification step (if any): Not applicable.
- Final dilution of a dissolved solid, stock liquid or gel: A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item within Arachis Oil BP.
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid formulations in vehicle, of the solid test item.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): Not applicable.

OTHER SPECIFICS: Not applicable.

Test animals

Species:
rat
Strain:
other: Han Wistar: RccHan: WIST
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: (P) Males 70 to 76 days (ca. 10 wks) Females 77 to 83 days (ca. 10 to 11 wks)
- Weight at study initiation: (P) Males: 270 - 314 g; Females: 172 - 216 g
- Fasting period before study: No.
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing. Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week. Cage distribution was randomised. The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups. Environmental enrichment: Aspen chew block provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary. Plastic shelter provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.
- Use of restrainers for preventing ingestion (if dermal): Not applicable.
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Males: six days before commencement of treatment. Females: 20 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: Feed: SDS VRF1 Certified pelleted diet – batch numbers and certificates of analysis provided in the full study report. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: Males: From: 2016-01-06 To: 2016-03-04 / Females: From: 2016-01-20 To: 2016-03-21

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a suspension in Arachis oil BP. The stability and homogeneity of the test item formulations were determined. Formulations up to 200 mg/mL in Arachis oil BP was achieved during formulation analysis. Formulations were found to be stable in Arachis oil BP, when assessed following storage at ambient temperature (nominally 21°C) for up to two days, and refrigeration (nominally 2 - 8°C) for up to 15 days.

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Applicant assessment indicates: Arachis oil BP was considered as appropriate based on test item solubility.
- Concentration in vehicle: Samples of each formulation prepared for administration in Week 1 of treatment, Week 4 of treatment for males and on Day 12 of lactation for females were analyzed for achieved concentration of the test item (method of analysis provided in full study report). Arachis oil formulations was assessed and confirmed at nominal concentrations of 1 mg/mL and 200 mg/mL. Test formulations analyzed for the study indicated the test item concentration was within ±10% of nominal concentrations, confirming accurate formulation. The test item concentrations for each group are indicated in table 1.
- Amount of vehicle (if gavage): Treatment volume was 4 mL/kg for control (negative, untreated group) and all treatment groups with applicable test item concentrations per group.
- Purity: BP grade Arachis Oil
- Other: Dose-formulations were analysed during the study and were reported as with ± 10% applied limits.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The homogeneity and stability was confirmed in Arachis oil BP formulations at nominal concentrations of 1 mg/mL and 200 mg/mL , during ambient temperature (nominally 21°C) for up to two days and refrigerated (nominally 2-8°C) for up to 15 days.
- The analysis consisted of GC FID analysis with internal calibration (Tetradecane in acetone ca. 2 mg/mL) within a dedicated formulation analysis report attached to the full study report. Samples of Arachis oil BP were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations along with internal standard for the calibration standards. These were then subjected to analysis by GC FID. The analytical method was validated (details available within the full study report). With LOD = 1.0167 μg/mL and LOQ = 3.389 μg/mL; linearity = > 0.999 and repeatability (n=6) of < 2%. Accuracy and precision was confirmed and mean procedural recovery was 94.1% (n=5) at 1 mg/mL and 95.6% (n=5) at 200 mg/mL.
- Mean concentrations of dose-formulations analysed during the study were within ± 10% applied limits confirming accurate test item/vehicle formulation.
Details on mating procedure:
- M/F ratio per cage: 1:1 within the same treatment groups.
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: Ejected copulation plug and sperm in vaginal smear. Day 0 of gestation was when positive evidence of mating was detected (daily checks).
- Further matings after unsuccessful attempts: No.
- After successful mating each pregnant female was caged (how): housed individually in a cages comprised of a polycarbonate body with a stainless steel mesh lid.
- Any other deviations from standard protocol: No
Duration of treatment / exposure:
Males: Two weeks pre-pairing up to necropsy after minimum of five weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation.
Frequency of treatment:
Daily; at approximately the same time each day.
F1 generation were not dosed.
Duration of test:
- Age at mating of the mated animals in the study: F0 generation: Males: ca. 12 weeks ; Females: ca. 13 weeks. (i.e. after two weeks treatment).
No. of animals per sex per dose:
10 per sex per dose (10 male / 10 female)
F1 generation were not dosed.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a previously conducted 28-day toxicity study (Report number attached to and cited in the full study report). The dose levels selected for the study were the same as used in the previous study. No findings in which would preclude the use of the associated dose levels.
- Rationale for animal assignment (if not random): Randomly assigned

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (and during acclimatisation period, once daily).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Signs associated with dosing: Week 1: Daily ; Week 2: Weekly; For F0 females: then Day 0, 7, 14 and 20 in Gestation Phase and Day 1, 7 and 13 in Lactation Phase. Detailed physical examinations: Weekly (F0 males), once each week (F0 females), then Day 0, 7, 14 and 20 in Gestation Phase, Day 1, 7 and 13 in Lactation Phase.

BODY WEIGHT: Yes
- Time schedule for examinations: F0 males: before dosing, and weekly thereafter. Day of necropsy ; F0 females: before dosing, and weekly until pairing, then days 0, 3, 7, 10, 14, 17 and 20 after mating, and then day 1, 4, 7, 13 and 14 of lactation plus day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not applicable.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable.
- Other: Food consumption was monitored during the study: F0 males: weekly before pairing ; F0 females: weekly before pairing, from the day that treatment commenced. Days 0-2, 3-6, 7-9, 10-13, 14-16 and 17-19 after mating. Days 1-3, 4-6 and 7-13 of lactation. Mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable.
- Time schedule for examinations: Not applicable.

OTHER:
1, See ‘FOOD CONSUMPTION’ field.
2. THYROID HORMONE ANALYSIS: Yes
- Time schedule: Day 4 of age (F1 offspring, two females per litter (where possible). Pooled sample by litter) ; Day 13 of age: Day 13 of age (F1 offspring, two males and two females per litter (where possible) ; Termination (All F0 males and F0 females)
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]
Statistics:
All statistical analyses were carried out separately for males and females. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/foetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

The following data types were analyzed at each timepoint separately:
Body weight, using absolute weights and gains over appropriate study periods
Food consumption, over appropriate study periods during gestation and lactation
Litter size, survival indices and sex ratio
Ano-genital distance
Organ weights, both absolute and adjusted for terminal body weight

Comparisons were performed:
Group 1 vs 2, 3 and 4

Typical statistical analysis included:
parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937)
For all other comparisons the F1 approximate test was applied. Which included:
parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972)
If the F1 approximate test was significant, Dunnett's test (Dunnett 1955, 1964) was performed instead

Additional tests, included Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953), Wilcoxon rank sum tests (Wilcoxon 1945) and/or Shirley's test (Shirley 1977) and Steel's test (Steel 1959), as appropriate.

For litter size and survival indices, Fisher’s exact tests (Fisher 1973).

Sex ratio were analyzed equivalent to Lipsitz (Lipsitz 1991). Each treated group was compared to control using a Wald chi-square test.

For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied.
Indices:
Offspring viability indices including; post-implantation survival index, live birth index, viability index, lactation index and sex ratio.
Historical control data:
Historical control data was available.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 650 mg/kg bw/day dose level: chin rubbing and/or salivation for males and females and underactivity and unsteady muscle reaction were seen in some males and females between Days 1 and 6 of treatment. One male and one female showed a poor righting reflex on Day 4 of treatment. At 300 mg/kg/day chin rubbing was seen in males and females. In lactation, chin rubbing and salivation continued to affect some females. In addition, one female receiving 650 mg/kg/day (80) was observed to be underactive, struggling with dosing and lying prostrate on Day 5 or 6 of lactation.
At 300 mg/kg bw/day dose level: isolated incidences of chin rubbing for females receiving 300 mg/kg/day and three females salivating on Day 20 of gestation. Some females receiving the dose showed chin rubbing and salivation, in lactation.
One male (4M 34) receiving 650 mg/kg/day was observed to have limited use of the right hind limb on Days 4-9 of treatment and one female (2F 60) receiving 30 mg/kg/day was observed to have struggled with dosing on Day 14 after mating. These signs were considered incidental and unrelated to treatment.
There were no clinical signs at detailed physical examination associated with treatment of the test item.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths in the F0 generation.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The group mean body weights of males and females were similar across all groups at the start of treatment.
At 650 mg/kg bw/day dose level: the body weight gain of males was low when compared with that of the Controls in Week 1 but it was similar to that of the Controls in Weeks 2-5 of treatment. The body weight gain of females was slightly higher than Controls prior to pairing. No dose response was apparent, there were no consistent intergroup differences and no effect of treatment is inferred. There was no effect of treatment on body weight or body weight gains for females receiving the test item during gestation or lactation.
At 300 and 30 mg/kg bw/day dose levels: the body weight gains of males were similar to that of the Controls in Weeks 1, 3 and 4, marginally higher than that of the Control in Week 2 and increased in Week 5. No dose response was apparent and no effect of treatment is inferred. The body weight gain of females was slightly higher than Controls prior to pairing. No dose response was apparent, there were no consistent intergroup differences and no effect of treatment is inferred. There was no effect of treatment on body weight or body weight gains for females receiving the test item during gestation or lactation, with the exception of 30 mg/kg bw/day between Days 13 to 14 of lactation (after removal of the offspring) which was slightly lower than that of Controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 650 mg/kg bw/day dose level: the food consumption of males receiving the test item was lower than that of the Controls in Week 1 of treatment and marginally lower than that of Controls in Week 2. It was comparable to controls in weeks 2 to 5 of treatment. Females food consumption was also slightly lower than that of the Controls in Week 1 of treatment but similar to that of the Controls in Week 2 of treatment. Although was similar to that of Controls throughout gestation. It was slightly higher than that of control animals during lactation. This may be a result of the slightly larger litter size for females receiving the test item.
Food efficiency:
not examined
Description (incidence and severity):
See 'Food consumption'
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):
Organ weights for males terminated after 5 weeks of treatment and for females killed on Day 14 of lactation showed no adverse effects of treatment.
At 650 mg/kg bw/day dose level: the lower mean testes weight in the group did not attain statistical significance and reflected the atypically low testes weight for male 31 which had small testes.
Gross pathological findings:
no effects observed
Description (incidence and severity):
no effects observed
There were no treatment related changes seen macroscopically related to treatment in males terminated after 5 weeks of treatment, and females terminated on Day 14 of lactation.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment related changes seen in histopathology related to treatment in males terminated after 5 weeks of treatment, and females terminated on Day 14 of lactation.

See 'Reproductive Function: Sperm Measures:

At 650 mg/kg bw/day dose level: there was an incidental finding that was considered unrelated to treatment.

One control male and one male given 650 mg/kg/day had microscopic findings in the testes and epididymides. These were not considered to be related to treatment. The control male (number 4) had slight bilateral seminiferous tubular atrophy in the testes, slight reduction in spermatozoa and slight cell debris in the epididymal lumen.
The male treated with 650 mg/kg/day (number 31) had severe bilateral seminiferous tubular atrophy in the testes, severe reduction in the numbers of spermatozoa and slight cell debris in the lumen of the epididymides. These microscopic changes correlate with the macroscopic findings of small testes and small epididymides. The microscopic changes seen in the testes and epididymis of the male given 650 mg/kg/day (number 31) are not considered to be related to treatment as only one male was affected and all germ cell layers in the testes were atrophied. Extensive tubular atrophy, often involving complete loss of all germ cells from all tubules in one or both testes, can sometimes be seen as a background finding in young adult rats.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment related changes seen in histopathology related to treatment in males terminated after 5 weeks of treatment, and females terminated on Day 14 of lactation.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
THYROID HORMONE ANALYSIS
There was no clear effect of treatment for the test item at 30, 300 or 650 mg/kg/day on the thyroid hormone levels of F0 adult males at termination or F1
offspring on Day 13 of age. There was a high degree of inter-individual variability, with atypically high values seen for the following:
1. Female offspring from Litter 45 (Control) had atypically high plasma T4 on Day 13 of age (8.65 ng/mL)
2. 30 mg/kg bw/day - group 2M number 15 had atypically high plasma T4 at termination (57.8 ng/mL)
3. Control - group 1M number 3 had atypically high plasma TSH at termination (5490 pg/mL)
4. Female offspring from Litter 41 (Control) had atypically high plasma TSH on Day 13 of age (4140 pg/mL)
5. 300 mg/kg bw/day - group 3M number 30 had atypically high plasma TSH at termination (4920 pg/mL)

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
One Control female and one female in Group 4 (650 mg/kg bw/day) failed to litter. After mating they were found not to be pregnant.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Post implantation survival index was slightly higher than that of the Controls amongst the litters of females receiving 30, 300 or 650 mg/kg/day, resulting in a slightly higher total litter size on Day 1. No effect of treatment is inferred.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
At up to 650 mg/kg bw/day: litter size, offspring survival was not affected by treatment.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
At up to 650 mg/kg bw/day: gestation length and gestation index were unaffected by treatment.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
At up to 650 mg/kg bw/day: All females showed normal estrous cycles before treatment. Estrous cyclicity, pre-coital interval, fertility and mating performance were unaffected by treatment. Litter size, offspring survival was not affected by treatment.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 650 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested

Maternal abnormalities

Abnormalities:
no effects observed
Description (incidence and severity):
No treatment related Adverse Effects observed up to the maximum dose level

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Male and female offspring body weight on Day 1 of age was similar in all groups. Thereafter, the body weight gain of male and female offspring in the 650 mg/kg/day group was lower than that of the Controls (11 and 10% respectively), and of male and female offspring in the 30 and 300 mg/kg/day groups.
This was not considered adverse as this did not attain statistical significance and the mean live litter size in 650 mg/kg bw/day group was slightly higher than controls due to chance. Resulting in higher within-litter competition. This is supported by total growth within 650 mg/kg bw/day was similar to controls (173 g versus 167 g in controls).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
At up to 650 mg/kg bw/day: litter size, offspring survival and sex ratio was not clearly affected by treatment.
The sex ratio (expressed as % males) of the litters of females receiving the test item at 650 mg/kg bw/day was slightly higher than that of the Control, with 5/9 litters in the 650 mg/kg/day group having a sex ratio of >70%, compared 2/9 litters in the Control group and none in the 30 or 300 mg/kg/day group. Assessment of the potential mechanisms of an adverse effect on sex ratio was considered and discounted within the study. It was considered within the study that the slightly higher percentage of male offspring in the 650 mg/kg/day group was likely due to chance (some litters containing more than 70% males occurred in the Control group) and was unrelated to parental treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Post implantation survival index was slightly higher than that of the Controls amongst the litters of females receiving 30, 300 or 650 mg/kg/day, resulting in a slightly higher total litter size on Day 1. No effect of treatment is inferred.

Male and female offspring body weight on Day 1 of age was similar in all groups. Thereafter, the body weight gain of male and female offspring in the 650 mg/kg/day group was lower than that of the Controls (11 and 10% respectively), and of male and female offspring in the 30 and 300 mg/kg/day groups.
This was not considered adverse as this did not attain statistical significance and the mean live litter size in 650 mg/kg bw/day group was slightly higher than controls due to chance. Resulting in higher within-litter competition. This is supported by total growth within 650 mg/kg bw/day was similar to controls (173 g versus 167 g in controls).
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
Ano-genital distances for male or female offspring were unaffected by treatment. A check was performed to assess for the presence or absence of nipple/areolae for the male offspring; no nipples were found.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Macroscopic examination of offspring did not reveal any findings attributable to treatment.
Visceral malformations:
no effects observed
Description (incidence and severity):
Macroscopic examination of offspring did not reveal any findings attributable to treatment.
Other effects:
no effects observed
Description (incidence and severity):
The thyroids of offspring were examined for follicular cell hypertrophy and hyperplasia and no evidence of these changes was present. Follicular lumen size/amount of colloid was semi-quantitatively graded using a five-point scale. There were no treatment related differences in this finding.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 650 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity and for reproductive / developmental toxicity for males and females is considered to be 650 mg/kg body weight per day.
Executive summary:

The study was performed according the requirements of OECD TG 421 guideline under GLP conditions. The purpose of this study was to assess general systemic toxic potential in rats, including a screening test for reproductive / development effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item by oral gavage administration for a least four weeks in Wistar Han : RccHan : WIST rats. Three groups, each comprising ten male and ten female rats, received test item at doses of 30, 300 or 650 mg/kg/day. A control group of ten males and ten females was similarly dosed with vehicle alone (Arachis oil BP), at the same dose volume as the treated groups. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were terminated on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure, if it occurred was in utero or via the milk. The F1 offspring were killed on Day 13 of age. Clinical condition, body weight change, food consumption, estrous cycles, pre-coital intervals, mating performance, fertility, gestation length, thyroid hormone analysis, organ weight, and macroscopic pathology and histopathology investigations were undertaken. For all offspring, clinical condition, litter size and survival, sex ratio, ano-genital distance, body weight and macropathology were also assessed. Nipple counts were performed on male offspring at day 13. The mean concentrations of the test item in test formulations were analyzed for the study and were within ±10% of nominal concentrations, confirming accurate formulation. The test item was determined to be stable at ambient storage for 2 days and refrigerated storage for up to 15 days within arachis oil formulations. There was no clear effect of treatment with the test item at 30, 300 or 650 mg/kg/day on the thyroid hormone levels of adult males at termination or F1 offspring on Day 13 of age. There was no effect on clinical condition or survival of males or, of females prior to pairing and during lactation, oestrus cycles, mating performance, fertility, gestation length or index, offspring survival, clinical condition, ano-genital distance or nipple counts, or macroscopic or microscopic pathology changes. Group mean body weights of males and females were similar across all groups at the start of treatment. Body weight gain of males receiving the test item at 650 mg/kg/day was low when compared with that of the Controls in Week 1 but was similar to that of the Controls in Weeks 2-5 of treatment. Transient post dosing sign included chin rubbing/salivation, underactivity and unsteady muscle reaction at 650 mg/kg/day, and chin rubbing/salivation at 300 mg/kg/day. The food consumption of males and females receiving 650 mg/kg/day was slightly low during the first week of treatment. There was considered to be no effect of treatment on the offspring sex ratio or body weight/weight gain. Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity and for reproductive / developmental toxicity was regarded to be 650 mg/kg/day for males/females.

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