Santicizer P1400

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-06-15 to 2018-02-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

With an extension (3 m/f pups per litter to PND 70) to evaluate sexual maturation of F1 generation included.

Deviations:

yes

Remarks:

The deviations were considered to have not affected the integrity or validity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Deviations:

yes

Remarks:

These deviations were considered to have not affected the integrity or validity of the study.

Test guideline 3

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test, Office of Prevention, Pesticides and Toxic Substances EPA 712–C–00–368, (7101), July 2000.

Deviations:

yes

Remarks:

These deviations were considered to have not affected the integrity or validity of the study.

Principles of method if other than guideline:

The study was a modified OECD 422 study with extended F1 phase in which pups were followed from birth to sexual maturation (PND 70) in F1 pups.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIES: Rat, recognised by international guidelines as a recommended test system.

METHOD OF TEST ITEM ADMINISTRATION: Oral ingestion is a possible route of human exposure to the test item.

DOSE LEVELS: The dose levels were selected based on a dose-range-finding toxicity study in rats (Envigo Study S56026 1,2-Cyclohexanedicarboxylic Acid, 1butyl 2 (phenylmethyl) ester, CAS no.1200806-67-2: 14-day Repeated Dose Oral (Gavage) Range Finding Toxicity Study in the Rat).

HIGH DOSE: The high dose 600 mg/kg was based on mild signs of toxicity observed in animals at 1000 and 700 mg/kg in a 14-day dose-rangefinding study (Envigo DRF S56026), the clinical findings suggested that more prolonged dosing in the definitive study, together with the potentially greater sensitivity of the pregnant females, may exceed e maximum tolerated dose level and may result in significant effects on the ability of females to maintain offspring postpartum.

MID DOSE: The intermediate dose 300 mg/kg was intended to ensure that the interval between dose levels was within the limits prescribed in the OECD test guideline number 422 and might result in safe dose for exposure to the test item.

LOW DOSE: The low dose 150 mg/kg was intended to ensure that the interval between dose levels was within the limits prescribed in the OECD test guideline number 422 and was also above the cut-off for GHS classification as “toxic” according to the criteria for a 28-day repeat dose toxicity study.

Test material

Test material information

Constituent 1

Reference substance name:

benzyl butyl cis-cyclohexane-1,2-dicarboxylate

Cas Number:

1931129-39-3

Molecular formula:

C19H26O4

IUPAC Name:

benzyl butyl cis-cyclohexane-1,2-dicarboxylate

Test material form:

liquid

Details on test material:

Name of substance: 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester
Alternative names: benzyl butyl cis-cyclohexane-1,2-dicarboxylate; Santicizer® Platinum P1400
Batch Number: 5840
CAS Numbers: 1931129-39-3 (cis-isomer); 1200806-67-2
Purity: 99.609 % (GC)
Expiration date: 2021-MAR-04

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: supplied by Envigo (Shardlow, UK) on behalf of the Sponsor (Polyadd Limited, U.K.); Batch no. 0900
- Expiration date of the lot/batch: 2017-01-17
- Purity test date: 2015-10-22

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: No special storage conditions are needed. Store away from incompatible materials (strong oxidizing agents). Keep the material sealed to avoid contact with moisture. Avoid high temperature.
- Stability under storage conditions: not specified
- Stability under test conditions: stable when stored at room temperature for 24 hours and stored refrigerated for 7 days.
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The homogeneity and stability was confirmed for the test item in vehicle formulations at nominal concentrations of 3.75 and 250 mg/mL when stored at room temperature for 24 hours and stored refrigerated for 7 days.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Liquid colorless

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Hannover Wistar rats

Details on species / strain selection:

Species: Rat - Species recognized by international guidelines as a recommended test system.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Supplier (Charles River Laboratories, France); Breeder (Charles River Laboratories, Germany)
- Females (if applicable) nulliparous and non-pregnant: yes, females were nulliparous and nonpregnant
- Age at study initiation: 10-11 weeks (males and females)
- Weight at study initiation: Males: 350-394 g; Females: 215-257 g
- Fasting period before study: Not specified
- Housing: Cages with standard, granulated, S8-15 sawdust bedding (J. Rettenmaier & Söhne)
Premating period: 5 animals/cage - Makrolon cages-IV
Mating period: one male and one female/cage - Makrolon cages-I
Postmating: individually - Makrolon cages-I
- Diet (e.g. ad libitum): Pelleted standard Teklad 2014C rat/mouse maintenance diet ad libitum (supplied by Envigo RMS, S.L., batches no.: 010616MA, 012016MA and 022216MA, expiry dates: 02 October 2016, 16 October 2016 and 18 November 2016,respectively). Pelleted standard Teklad 2018C rat/mouse maintenance diet ad libitum(supplied by Envigo RMS, S.L., batch no.: 020916MA, expiry date: 5 November 2016), for lactating females and pups (until day 21 post partum).
- Water (e.g. ad libitum): Tap water in bottles ad libitum
- Acclimation period: 12 days prior to estrus cycle evaluation under test conditions

DETAILS OF FOOD AND WATER QUALITY: The diet was analyzed by the manufacturer for contaminants and to check its composition and the water underwent bacteriological, chemical and contaminant analysis. The results were included in the Annex 4 of the study report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23°C - monitored continuously
- Humidity (%): 40-70%
- Air changes (per hr): 15-20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light (artificial fluorescent light)

ENVIRONMENTAL ENRICHMENT
- different types of material specific to the species (e.g. paper wool and sizzlenest) were provided to reduce stress, enhance well-being and improve behaviour.

IN-LIFE DATES: From: 2016-06-15 To: 2017-10-26

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Oral route selected since oral ingestion is a possible route of human exposure to the test material. Test material was administered orally via gastric gavage using glass syringes.

PREPARATION OF DOSING SOLUTIONS: The appropriate amount of test material was added in a suitable glass container followed by the vehicle to reach the final concentration. The vial was gently inverted for a minimum five times to mix the formulation completely. This solution was aliquoted for each administration day in glass containers.

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil (justification not specified)

- Concentration in vehicle: The amount of test material to be administered was calculated according to the most recently recorded body weight.
- Amount of vehicle (if gavage): 4 mL/Kg body weight
- Lot/batch no. (if required): Sigma-Aldrich Reference MKBV2080V and MKBW9504V

Details on mating procedure:

- M/F ratio per cage: 1:1
> Premating period (5 animals/cage) Makrolon cages -IV
> Mating period (one male and one female/cage) Makrolon cages -
> I Postmating (individually) Makrolon cages -I

- Length of cohabitation:
> During the mating period the females were housed with males within each dose group (one male:one female) until evidence of copulation was observed for a period of 18 or 24 hours.
> The females were removed and housed individually when:
a) The daily vaginal smear was sperm-positive, and/or
b) A copulation plug was observed. The day of mating evidence was designated day 0 post coitum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For each analysis occasion, freshly prepared test formulations were analyzed in accordance with the analytical procedure. Samples were disposed of once satisfactory results were achieved. The formulations received were diluted with tetrahydrofuran. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with tetrahydrofuran and then shaken to dissolve. Where necessary, sample solutions were further diluted with tetrahydrofuran to achieve the working concentration.

The dose formulation concentration was determined twice during the study and once (top/middle/bottom) for homogeneity in samples taken from the formulation to be administered to Groups 2 to 4. One additional sample was also taken from the vehicle prepared. The formulations were quantified by gas chromatography according to the analytical procedure validated in Study YR67PH (DRF). The homogeneity and concentration of the formulations was analysed prior to the first day of dosing with the 1st analysis and concentration with the 2nd analysis. On both occasions, duplicate samples of the dosing solution (approx. 5 mL each) were transferred to labelled vials. Aliquot 1 was used for analysis and was shipped refrigerated in the dark to the PI (Paul Watson, Envigo Shardlow, UK). This aliquot was discarded after analysis. Aliquot-2 samples will be retained for any possible subsequent needs at Envigo CRS, S.A.U. and will be discarded after issuing the final phase or the final report unless otherwise requested by the Sponsor. Each sample was labelled at least with study number, phase number (YR67HP), aliquot number, treatment, concentration, formulation preparation date, formulation administration date and storage conditions. The test item was used as analytical
standard.

Duration of treatment / exposure:

Parental generation: starting two weeks before mating through a minimum of 28 days for males (after confirming female pregnancy) and until day 20 postpartum for females including the day before sacrifice.

Pups for sexual maturation from day 21 postpartum until sexual maturation 70 days postpartum (although direct treatment starts at or soon after weaning, all offspring have potential indirect exposure in utero and through the milk during lactation). Remaining pups (sacrificed on day 4 or 13 postpartum): not treated. Potential indirect exposure in
uteroand through the milk during lactation.

Frequency of treatment:

Once daily

Details on study schedule:

Parental generation: starting two weeks before mating through a minimum of 28 days for males (after confirming female pregnancy) and until day 20 postpartum for females including the day before sacrifice.

Pups for sexual maturation from day 21 postpartum until sexual maturation 70 days postpartum (although direct treatment starts at or soon after weaning, all offspring have potential indirect exposure in utero and through the milk during lactation).

Remaining pups (sacrificed on day 4 or 13 postpartum): not treated. Potential indirect exposure in utero and through the milk during lactation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

12/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on a dose-range-finding (DRF) toxicity study in rats (Envigo Study S56026: 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2 (phenylmethyl) ester, CAS no.1200806-67-2: 14-day Repeated Dose Oral (Gavage) Range Finding Toxicity Study in the Rat).

The low dose 150 mg/Kg was intended to ensure that the interval between dose levels was within the limits prescribed in the OECD test guideline number 422 and was also above the cut-off for GHS classification as “toxic” according to the criteria for a 28-day repeat dose toxicity study. The intermediate dose 300 mg/Kg was intended to ensure that the interval between dose levels was within the limits prescribed in the OECD test guideline number 422 and might result in safe dose for exposure to the test material. The high dose 600 mg/Kg was based on mild signs of toxicity observed in animals at 1000 and 700 mg/Kg in the 14-day DRF study (Envigo DRF S56026). The clinical findings
suggested that more prolonged dosing in the definitive study, together with the potentially greater sensitivity of the pregnant females, may exceed the maximum tolerated dose level and may result in significant effects on the ability of females to maintain offspring postpartum.

- Rationale for animal assignment (if not random): Animals were allocated at random based on mean body weight similarity among groups. Females were evaluated pre-exposure for estrus cyclicity.

- Fasting period before blood sampling for clinical biochemistry: Yes, animals were fasted overnight before blood sampling but allowed ad libitum access to water. The samples were collected early in the morning.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality was checked for twice daily. Any rat sacrificed during the study was subjected to macroscopic examination.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations:
P males: at pretest, on first day of dosing and twice weekly during the pre-pairing until sacrifice day (included).
P females: at pretest, on first day of dosing, twice weekly during the pre-pairing and pairing period and on days 0, 4, 7, 11, 14, 17 and 20 of pregnancy and within 24 hours of parturition (day 0 or 1 postpartum) and on days 4, 7, 14, 17 and 21 (as terminal body weight).

Pups for sexual maturation: twice weekly after weaning

Body weight during mating period is not reported.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
P males: at pretest, once weekly during the pre-pairing and weekly during pre-treatment, treatment and postpairing period.
P females: at pretest, once weekly during the pre-pairing and treatment periods and days 0-7, 7-14, 14-20 post coitum and days 1-7 and 7-14 postpartum.
Pups for sexual maturation: once weekly
Food consumption was not examined during the mating period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Males: following at least 28 administrations of the test item/vehicle (on day of sacrifice)
Females: on day 21 postpartum (on day of sacrifice)
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia)
- Animals fasted: Yes (The animals were fasted overnight before blood sampling but allowed ad libitum access to water. The samples were collected early in the morning)
- How many animals: five animals/sex/ group
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Males: following at least 28 administrations of the test item/vehicle (on day of sacrifice)
Females: on day 21 postpartum (on day of sacrifice)
- Animals fasted: Yes (The animals were fasted overnight before blood sampling but allowed ad libitum access to water. The samples were collected early in the morning)
- How many animals: five animals/sex/ group
- Parameters checked in table [No.3] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: before the first exposure to the test item, once weekly thereafter and one day before sacrifice. These observations were performed outside the home cage, in a standard arena, at least one hour after dosing (where applicable) to ensure identification of any transient effects
- Dose groups that were examined: performed on all test and control group animals
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

Grip strength assessment:
Grip strength (fore- and hind limbs) was measured in five randomly selected males per group once during the final week of treatment and at least one hour after dosing. Five randomly selected females per group were similarly evaluated on day 20 postpartum.

Motor activity assessment:
Motor activity was measured in the animals mentioned above with an activity motor system (AMS from Medical Instruments GmbH (FMI) and DeMeTec-Ams version 2.0 GmbH). Activity of the animals was recorded in 5-minute intervals over a 30-minute period and measured quantitatively (beam counts).

Sensory reactivity assessment:
Sensory reactivity to different stimuli was evaluated in the animals mentioned above. Details presented in Table No. 4.

IMMUNOLOGY: No

OTHER:
Thyroid Hormones: Blood samples were taken based on the following schedule:
• from all two pups per litter on day 4 after birth,
• from all pups per litter on day 13 after birth,
• from all surviving adult males, at termination,
• from all surviving dams on day 21 postpartum, and
• from cohort F1 on PND 70

All samples were stored under appropriate conditions after centrifugation (at 4 ºC and 2000 g for 10 minutes) to obtain serum. Samples from the PND 13 and 70 pups and adults were assessed for plasma levels of TSH and also serum for T4 in adult animals. Samples were shipped frozen to the PI (Envigo CRS Limited Test Site, Huntingdon) for analysis. Further assessment of T4 in serum samples from the adult animals at sacrifice and fromday-4 pups was done when relevant. Pup samples can be pooled by litter for thyroid hormone analyses. If not, an evaluation of potential effects on the thyroid function could be done in F1 offspring. This would comprise detailed microscopic examination of the thyroid glands from Day-13 offspring together with evaluation of potential effects upon plasma TSH levels in Day-4 and Day-13 offspring. Samples specifically intended for hormone determination were obtained at a comparable time of the day.

Analysis of serum samples for T4 was conducted at Envigo CRS Limited (Huntingdon) under the supervision of a Principal Investigator (Hariharasudhan Bose) for this study phase. Samples were analyzed using LC-MS/MS instrumentation, based on the bioanalytical method BM/2016/0632 (V
alidation study number: FF58YR).

Thyroid Hormones Acceptance Criteria
Since thyroid hormones are endogenous compounds, the acceptance criteria were widened. The accuracy (RE) determined at each QC concentration level was within ±20% (25% for LLOQ). The RE of at least 67% of the QC samples overall within the batch was within ±20% of their nominal concentration, including at least 50% at each concentration level. At least 75% of the calibration standards were within ±20% of the nominal concentration (±25% at LLOQ). When diluted test samples wereincluded in the batch, then 50% of the diluted QC samples in the batch (normally n = 2) were within 20% of their respective nominal concentration for the diluted sample data to be accepted.

Oestrous cyclicity (parental animals):

SPECIALIST EVALUATION P GENERATION
- Stage of oestrus:
Stage of estrus Vaginal smears for all females were collected for evaluation of oestrus cycle two weeks before treatment start to select female s with regular cycle. For the study Smearing of individual females was monitored daily from start of treatment until evidence of mating. Vaginal s mears were examined on the day of necropsy to determine the stage of the estrous cycle and allow correlation with histopathology of female re productive organs.

- Mating:
On day 15 of treatment, the mating period started while the test item was still being administered. During the mating period the females were ho used with males within each dose group (one male:one female) until evidence of copulation was observed for a period of 18 or 24 hours.
The females were removed and housed individually when:
a) The daily vaginal smear was sperm-positive, and/or
b) A copulation plug was observed. The day of mating evidence was designated day 0 post coitum.

- Parturition
From day 20 post coitum to 24 post coitum, the females were examined three times daily for signs of parturition. The duration of gestation (days) was recorded. The day of birth (completion of parturition) is defined as day 0 postpartum.

- Nursing:
The females that gave birth were checked twice daily, together with the litter status check, to observe whether they nursed their offspring.

Sperm parameters (parental animals):

A qualitative staging of spermatogenesis and histopathology evaluation of interstitial cells of all males from the control and high-dose groups was performed by the histopathologist.

Litter observations:

- Check at delivery:
>The number of stillbirths and of live and dead pups and any macroscopic anomalies were recorded for each litter within 24 hours of parturition (day 0 or 1 postpartum).
>If parturition ended before 7 am, this day was considered as day 1 postpartum and if parturition ended after 7 am this day was considered as day 0 postpartum.
>Pups were sexed, counted, weighed, identified with a pen and externally examined.

- Litter status Twice daily.
>Any pup sacrificed or found dead during the study was subjected to macroscopic examination.

- Litter clinical signs
> Pups were observed once daily.

- Litter sex:
> Days 1, 4, 13 and 21 postpartum

- Litter body weight:
> Days 1, 4, 7 and 13.
> Pups used for sexual maturation (3/sex/litter) were weighed on day 21 postpartum (PND) and then weekly until day 70 (included).

- Litter behavior test Day 1 postpartum:
>surface righting reflex

- Litter size standardization:
> On day 4 after birth, the size of each litter was adjusted by eliminating extra pups, as nearly as possible, to four pups per sex per litter. Pups were identified by finger tattoo.
>Ano-genital distance (AGD)
> The AGD of each pup was measured on PND 4 before size standardization. The distance between the genital papilla and anus was measured using a non-rigid caliper. Pup body weight was collected on the day the AGD was measured and the AGD was normalized to a measure of pup size, preferably the cube root of body weight.

Nipples/areolae:
>The number of nipples/areolae in male pups was counted on PND 13

Sexual maturation
>On day 13 after birth, three males and three females were selected at random and the size of each litter was adjusted by eliminating extra pups, when possible.
>Males: Examined daily from Day 38 of age for the completion of balano-preputial separation. Body weight was recorded on day of completion of separation.
>Females: Examined daily from Day 25 of age until vaginal opening occurred. Body weight was recorded on day of vaginal opening.

Postmortem examinations (parental animals):

SACRIFICE
Animals sacrificed in extremis were necropsied. Their organs were extracted and fixed but not weighed.

PARENTAL FEMALES
Females on day 21 postpartum and those showing total litter loss were deeply anesthetized with sodium pentobarbital administered intraperitoneally, exsanguinated by excision of the axillary vessels and aorta, and necropsied. The mated females that did not give birth or show signs of pregnancy were sacrificed 24-26 days post coitum as they were not mated again.

The postmortem examination of the external surface of the body, all orifices, cranial, thoracic and abdominal cavities and their contents with emphasis on the uterus, number of implantation sites was performed. The observation of the organs in situ and description of all macroscopic abnormalities are recorded.
A vaginal smear was examined to determine the stage of estrus cycle.

When no implantation site was evident, the uterus was placed in an aqueous solution of ammonium sulfide (Salewski, 1964) to accentuate possible hemorrhagic areas of implantation sites

PARENTAL MALES
After at least 29 administrations, all males were deeply anesthetized with sodium pentobarbital administered intraperitoneally, then exsanguinated by excision of the axillary vessels and aorta and necropsied subjected to confirmation of successful mating. The postmortem examination of the external surface of the body, all orifices, cranial, thoracic and abdominal cavities and their contents was performed. The observation of the organs in situ and description of all macroscopic abnormalities was recorded.

PATHOLOGY
The organs to be weighed (Table No. 5) were recorded on the scheduled necropsy dates for all surviving parental animals and group mean and adjusted values were calculated. Samples of tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution (10% formalin) unless otherwise indicated as well as specimens of abnormal tissue.

Reproductive organs of all control and high dose animals as well as of animals selected for sexual maturation were also examined

All organ and tissue samples (except for the nose) of selected animals from groups 1 and 4 (5 animals/sex, as indicated in the actual pathology report) to be examined were processed, embedded, cut at an approximate thickness of 2-4 micrometers and stained with hematoxylin and eosin. Special stains were used at the discretion of the study pathologist. The bone marrow smears were stained using the May Grünwald-Giemsa method and kept at the test facility for possible further investigation.

HISTOLOGY
Slides of organs and tissues collected at necropsies of all animals to be evaluated, except for the bone marrow smear for which no test was planned, were examined by the pathologist at Envigo CRS, S.A.U. A description of all abnormalities is included in the report. Where possible, the microscopic findings were correlated with the gross observations. As test-item-related morphological changes were detected in thyroids in high-dose animals. The thyroid glands were examined in all adult animals from Group 2 and 3, as well as on day 70 PND from cohort F1.

A histopathology evaluation of interstitial cells of all males from the control and high-dose groups was performed by the histopathologist

Postmortem examinations (offspring):

SACRIFICE
On days 4 and 13 postpartum, selected pups were sacrificed by intraperitoneal injection of sodium pentobarbital and subjected to macroscopic examination. On day 70 postpartum, pups selected for evaluation of sexual maturation postpartum were also sacrificed by intraperitoneal injection of sodium pentobarbital and subjected to macroscopic examination.

NECROPSY
Offspring that died during the study and female pup no. 525 sacrificed for welfare reasons were examined externally and necropsied, except those excessively cannibalized or autolytic. Necropsy and a macroscopic examination were carried out on all other animals. Samples of organs and tissues with macroscopic alterations were taken and preserved in neutral phosphate buffered 4% formaldehyde solution for possible microscopic examination. A vaginal smear was taken to determine the stage of oestrus cycle, if considered necessary.

Statistics:

Please see the section 'Any other information on materials and methods incl. tables' for information on statistics.

Reproductive indices:

Mating Performance and Fertility
- Pre-coital time: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
- Fertility Indices: The following indices were calculated for each group:

1) Percentage Mating (%) = (Number of females mated / Number of females paired) x 100
2) Conception rate (%) = (Number of females achieving pregnancy / Number of females mated) x 100
3) Fertility index (%) = (Number of females achieving pregnancy / Number of females paired) x 100

- Implantation Losses (%): Group mean percentile pre-implantation and post-implantation loss was calculated for each female/litter as follows:
1) Pre–implantation loss = ((Number of corpora lutea - number of implantation sites) / (Number of corpora lutea)) x 100
2) Post–implantation loss = ((Number of implantation sites - Total number of offspring born) / (Number of implantation sites)) x 100

Gestation and Parturition Data: The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.
- Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
- Gestation Index:
Gestation Index (%) = (Number of females with living pups / Number of females pregnant) x 100

Offspring viability indices:

Litter Responses: values were first calculated for each litter and the group mean was calculated using the individual litter values. Group mean values included all litters reared to termination (Day 4 of age).

1) Post–natal loss = (Number of offspring / Number of offspring alive on Day 4) x 100

2) Viability Index (%) = (Number of alive pups on Day 4 / Number of offspring alive on Day 1) x 100

3) Live birth index (%) = (Number of offspring alive on Day 1 / Number of offspring delivered) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs considered to indicate an immediate or delayed reaction to treatment.

Salivation was observed during postnatal periods in males at 300 and 600 mg/Kg/day as well as during the treatment period at 600 mg/Kg/day. In females, salivation was observed in the same treatment groups during treatment, mating, gestation and lactation periods, as well as occasionally in females receiving 150 mg/Kg/day during gestation and lactation.

Although this was considered a test-item related effect, it could be related to palatability and thus not considered to be an adverse effect, as the animals could taste the test substance when the probe (used for gavage) was removed after administration.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no test material-related deaths.

Male no. 66 administered the test item at 600 mg/Kg/day was sacrificed on day 14 of treatment for animal welfare reasons, as there was a problem during the administration handling the previous day (the handler accidently lost hold of the animal as it tried to escape and it fell to the floor, after which it did not recover).

Females no. 109 (0 mg/Kg/day) and 161 (600 mg/Kg/day) were sacrificed for animal welfare reasons due to difficulties during parturition. As females were sacrificed, the corresponding litters were also killed and examined macroscopically:

- In the case of female no. 109, the cause for the poor condition was hematoma/hemorrhage, which most likely explains the sudden pallor displayed by that animal. Hysterectomy was performed and the foetuses (one male and one female in left uterine horn) that were found deadwere examined macroscopically, showingadvanced autolysis in abdominal and thoracic cavities.

- In female no. 161, 4 males and 4 females were alive when the hysterectomy was performed and 5 males and 2 females were found dead. No alterations were observed in their necropsy.

Females no. 102 (0 mg/Kg/day) and 149 (600 mg/Kg/day) were sacrificed on days 2 and 3 of lactation, respectively, due to total litter loss.

On day 24 of gestation, female no. 106 (0 mg/Kg/day) was sacrificed as no signs of parturition were observed. At necropsy, 1 implantation was observed in left uterine horn and consequently, this female was considered to be pregnant.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect on body weights during the study.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no indication of an effect on food consumption during the treatment period.

The statistically significant increases noted at 300 and 600 mg/kg/day in males (postnatal days 8 and 15) or in females at 600 mg/kg/day on gestation day 14 were not considered to have been toxicologically relevant given theirmagnitude (no more than 1.2 times the Control mean values), as it was transient and as there were no effects in body weights that could be associated with it.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no changes of toxicological relevance.

Males receiving 600 mg/kg/day showed significantly higher mean neutrophil and basophil values compared to the Control group. This tendency was also observed in females receiving 600 mg/kg/day but it was not statistically significant. There was no dose-effect relationship. Females on day 21 of lactation showed no other differences from the Control group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Blood chemistry examination performed on the day of sacrifice in males revealed a dose-related incre ase in protein and globulin values in males. These differences were statistically significant at 300 and 600 mg/kg/day when compared to the Control group. However, this increase in protein and globulin values was not observed in females.

No other differences were observed when comparing the test item administered groups with the Control group.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Motor activity was considered not to be affected by the test item. Statistically significant increases observed in females at 600 mg/Kg/day (compared to Control) at 5 minutes were not considered relevant or test material-related, as they were occasional and there was no similar difference observed in males.

There was no indication of any adverse effects of the test item on sensory reactivity or grip strength assessments.

Given the magnitude (from 9 to 11 % versus Control) and the lack of a dose-effect relationship, the statistically significant differences observed at 300 and 600 mg/kg/day in the mean forelimb grip strength of femalesand at 600 mg/kg/day in the mean forelimb and hind limbs (11%) were not considered to be toxicologically relevant.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no histopathological correlates for the increased liver weight in F0 males treated at the high dose.

In the kidneys of some F0 males treated with the high dose, increased amounts of hyaline droplets were seen. This correlates with the increased kidney weight in this group. However, the level of hyaline droplet accumulation observed was within the normal limits in this species and strain and was therefore not considered to be related to treatment.

In the testes, no cell or stage specific abnormalities were noted in males treated at 600 mg/kg/day. Interstitial cells were also assessed qualitatively, and no alterations were seen in treated males.

In the thyroid glands, minimal hypertrophy of the follicular epithelium was observed in some males of all treated groups, with a dose-effect relationship. It was also observed in isolated females of all treated groups.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

There was a minimal or slight hypertrophy of the follicular epithelium of the thyroid glands in males and females of all treated groups (150, 300 and 600 mg/kg/day). This finding could be considered to be within the normal variation in females at 150 and 300 mg/kg/day due to the low incidence recorded (2/12 at 150 mg/kg/day and 1/11 at 300 mg/kg/day), and the lack of effects in the T4 determinations. The decrease observed in T4 in F0 males at 600 mg/kg/day also correlates with the histopathological findings.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No effect was observed in the length of the estrous cycle

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

In the testes, no cell or stage specific abnormalities were noted in males treated at 600 mg/kg/day. Interstitial cells were also assessed qualitatively, and no alterations were seen in treated males.

Reproductive performance:

no effects observed

Description (incidence and severity):

No effect was observed on pre-coital interval, mating performance, fertility or gestation length or index when compared to the control group. No cell or stage specific abnormalities were noted in males treated at 600 mg/kg/day. Interstitial cells were also assessed qualitatively, and no alterations were seen in treated males either.

Details on results (P0)

Mortality: There were no test-item-related deaths.

Clinical Signs: There were no clinical signs considered to indicate an immediate or delayed reaction to treatment.

Food Consumption: There was no indication of an effect on food consumption during the study.

Body Weight: There was no effect on body weights during the study.

FOB: Motor Activity: Motor activity was considered not to be affected by the test item.

FOB: Sensory Reactivity and Grip Strength: There was no indication of any adverse effects of the test item on sensory reactivity or grip strength assessments.

Hematology and Coagulation: There were no changes of toxicological relevance.

Clinical Biochemistry: Blood chemistry examination performed on the day of sacrifice in males revealed a dose-related increase in protein and globulin values in males. These differences were statistically significant at 300 and 600 mg/kg/day when compared to the Control group. However, this increase in protein and globulin values was not observed in females.

Macropathology: The macroscopic examination performed on day 21 postpartum in females and after at least 28 days of treatment in males revealed no test-item-related lesions. All findings were considered to be incidental and unrelated to treatment with the test item.

Organ Weights: A dose-related increase was recorded in kidney weights in all male treatment groups (maximum effect was observed in adjusted mean values at 600 mg/kg/day and was 17% higher versus Control). In addition, mean adjusted liver and spleen weights in males at 600 mg/kg/day were 40 and 22% higher, respectively, than those recorded in the Control group. In males, mean adjusted thymus weight was 18% lower at 600 mg/kg/day when compared to the Control group. All these differences were statistically significant.
Dose-related decreases in uterus, cervix and oviduct weights were observed (maximum effect 23% in adjusted mean values). Statistically significant differences were observed at 600 mg/kg/day.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: minimal to slight microscopic changes in the thyroid glands

Remarks on result:

other: At 600 mg/kg/day histological findings observed in males and females (minimal to slight microscopic changes in the thyroid glands) were considered non-adverse in the absence of consistent changes in hormone levels .

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Mortality:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed which were indicative of a reaction to the test item.

Female no. 525, administered the test item at 300 mg/kg/day, was sacrificed on day 18 of treatment phase. This female showed stereotypic movements (cycling) and prostration. Given the incidence and the lack of similar observations at 600 mg/kg/day, it cannot be considered a test-item-related effect.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no test-item-related deaths.

Female no. 525, administered the test item at 300 mg/kg/day, was sacrificed for welfare reasons, given the clinical signs observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no differences compared to the Control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no indication of an effect on food consumption during the treatment period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

TSH ANALYSIS
Low mean values (statistically significant differences at 300 and 600 mg/kg/day) were recorded in offspring (pooled samples including males and females) on day 4 when compared to Control (53 and 60%, respectively). At 600 mg/kg/day, statistically significant high mean values were observed on PND 70 in males and females compared to Control (31 and 55%, respectively).

T4 ANALYSIS
In the T4 analysis, F0 males and PND 70 males administered the test item at 600 mg/kg/day showed mean serum T4 concentrations slightly lower than those of the Control group (16% and 8%, respectively). These differences were statistically significant. In addition, significant differences were also observed at 600 mg/kg/day in offspring on day 4 (low mean values, 19% with respect to Control).

Clinical biochemistry findings:

not specified

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

No adverse treatment-related effects were observed in the reproductive organs relating to alterations in the sexual maturation of male or female F1 pups.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There were no differences between the test-item-administered groups and Control animals.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No test-item effect was observed in nipples areolae in male pups.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no differences in the mean weights of the thyroids or parathyroids in the test-item-administered animals compared to the Control group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The thymus discoloration observed (Control Group: 1 male and 2 females, 150 mg/kg/day: 2 females and at 300 mg/kg/day: 1 female) was considered incidental and unrelated to treatment due to the distribution of this finding. No other macroscopic alterations were observed among the study animals.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All histological findings were considered to be incidental and unrelated to the test item.

A dose-related increase in incidence and/or severity of follicular cell hypertrophy in thyroid glands was seen in the test-item-administered males when compared with controls. Minimal hypertrophy of the follicular epithelium was observed in some females at 600 mg/kg/day, and was considered to represent normal variation in follicular epithelial height in juvenile rats, rather than a treatment effect.

Other effects:

no effects observed

Description (incidence and severity):

Check at Delivery / Litter Size and Sex:
- There was no effect of the test item on survival, litter size or sex ratio. There were no differences in the percentage of viable pups or pre/post-natal losses in the test item administered groups when compared with the Control group on days 4, 13 or 21.

Developmental Filial Data: Behavior, Nipples, Areolae and Sexual Maturation
- There were no differences in the surface righting test between the animals administered the test item and those of the Control group. In addition, no test-item effect was observed in balanopreputial separation or nipples areolae in males or in vaginal opening in females.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive and Developmental Toxicity

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 6. F0 Food consumption - group mean values (g/animal/day): Males
Dose Group		Pre-treatment Period		Treatment Period		Postnatal Period
		Day 8	Day 12	Day 8	Day 15	Day 8	Day 15
Statistical Test		Av	Av	Wi	Wi	Wi	Wi
Males
Control	Mean	26	26	21	20	18	19
	SD	3.7	3.2	0.5	0.3	1.2	1.1
	N	3	3	3	3	12	12
150 mg/Kg/day	Mean	25	25	21	20	18	18
	SD	2.4	3.3	0.7	0.4	1.9	1.4
	N	3	3	3	3	12	12
300 mg/Kg/day	Mean	25	25	22	22	20**	21*
	SD	3.0	3.2	1.0	0.8	1.8	1.7
	N	3	3	3	3	12	12
600 mg/Kg/day	Mean	38	39	22	22	22**	21**
	SD	20.0	20.5	1.2	1.6	2.0	2.5
	N	2	2	3	3	11	11

Av: Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests where appropriate

Wi: Treated groups compared with Control using Williams’ test

* p≤0.05

** p≤0.01

Table 7. F0 Food consumption - group mean values (g/animal/day): Females
Dose Group		Pre-treatment Period		Treatment Period		Gestation Period			Lactation Period
		Day 8	Day 12	Day 8	Day 15	Day 7	Day 14	Day 20	Day 7	Day 14
Statistical Test		Av	Av	Wi	Wi	Wi	Wi	Wi	Sh	Sh
Females
Control	Mean	22	24	16	17	18	21	21	27	48
	SD	4.3	5.5	0.8	0.5	2.0	2.0	2.4	6.7	10.9
	N	3	3	3	3	12	12	12	9	9
150 mg/Kg/day	Mean	19	19	16	16	19	21	20	30	52
	SD	3.0	2.9	0.2	0.1	2.3	1.6	2.3	4.7	4.2
	N	3	3	3	3	12	12	12	12	12
300 mg/Kg/day	Mean	24	24	16	16	19	20	22	42	51
	SD	5.2	5.5	0.2	0.3	2.3	2.2	2.8	31.3	4.9
	N	3	3	3	3	12	12	12	12	11
600 mg/Kg/day	Mean	17	18	16	16	20	22*	23	27	45
	SD	0.5	1.0	0.3	0.6	2.1	1.9	1.8	16.5	13.1
	N	2	2	3	3	3	12	12	11	11

Av: Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests where appropriate

Sh: Treated groups compared with Control using Shirley’s test

Wi: Treated groups compared with Control using Williams’ test

* p≤0.05

** p≤0.01

Table 8. F0 Body weight - group mean values - Males
Period	Day (Statistical Test)	Dose group:	Control	150 mg/Kg/day	300 mg/Kg/day	600 mg/Kg/day
Pre-treatment Period	Day 2 (Av)	Mean	344.6	343.6	344.3	346.7
		SD	9.36	7.72	11.98	11.54
		N	12	12	12	12
		% of control	-	100	100	101
	Day 12 (Av)	Mean	369.5	369.8	368.0	370.6
		SD	10.59	11.03	12.42	10.85
		N	12	12	12	12
		% of control	-	100	100	100
Treatment Period	Day 1 (Wi)	Mean	371.7	372.8	371.9	373.8
		SD	11.09	12.56	12.82	11.41
		N	12	12	12	12
		% of control	-	100	100	101
	Day 4 (Wi)	Mean	374.2	378.3	375.7	377.3
		SD	12.75	12.97	13.18	12.74
		N	12	12	12	12
		% of control	-	101	100	101
	Day 8 (Wi)	Mean	382.2	384.6	381.7	385.3
		SD	14.08	14.81	13.76	12.09
		N	12	12	12	12
		% of control	-	101	100	101
	Day 11 (Wi)	Mean	384.7	383.4	385.0	381.9
		SD	16.26	15.87	12.25	10.93
		N	12	12	12	12
		% of control	-	100	100	99
	Day 14 (Wi)	Mean	390.0	391.0	390.4	391.9
		SD	16.78	16.35	13.85	13.06
		N	12	12	12	12
		% of control	-	100	100	100
Postnatal Period	Day 1 (Wi)	Mean	387.1	386.2	386.2	387.5
		SD	17.00	17.15	13.88	12.92
		N	12	12	12	11
		% of control	-	100	100	100
	Day 3 (Wi)	Mean	389.4	389.8	391.2	392.9
		SD	13.95	16.33	13.35	13.17
		N	12	12	12	11
		% of control	-	100	100	101
	Day 7 (Wi)	Mean	391.8	391.7	394.3	396.1
		SD	16.14	17.20	14.00	10.92
		N	12	12	12	11
		% of control	-	100	101	101
	Day 10 (Wi)	Mean	395.6	394.0	397.9	399.6
		SD	18.07	17.24	14.59	10.74
		N	12	12	12	11
		% of control	-	100	101	101
	Day 14 (Wi)	Mean	398.4	396.5	399.8	399.5
		SD	18.75	17.84	14.60	16.48
		N	12	12	12	11
		% of control	-	100	100	100
	Day 17 (Wi)	Mean	398.8	404.3	402.7	396.0
		SD	19.51	23.90	15.19	19.54
		N	7	6	7	6
		% of control	-	101	101	99
	Day 21 (Wi)	Mean	395.8	405.6	400.6	400.1
		SD	24.03	22.68	17.35	20.0
		N	6	6	6	6
		% of control	-	102	101	101
	Day 24 (Wi)	Mean	398.8	407.3	402.0	400.1
		SD	24.72	21.76	18.01	20.62
		N	6	6	6	6
		% of control	-	102	101	100
	Day 28 (Tt)	Mean	396.7	-	399.8	-
		SD	38.47	-	-	-
		N	2	-	1	-
		% of control	-	-	101	-

Table 9. F0 Body weight - group mean values - Females
Period	Day (Statistical Test)	Dose group:	Control	150 mg/Kg/day	300 mg/Kg/day	600 mg/Kg/day
Pre-treatment Period	Day 2 (Av)	Mean	228.3	227.2	226.4	225.3
		SD	10.21	8.61	9.79	10.01
		N	12	12	12	12
		% of control	-	100	99	99
	Day 12 (Av)	Mean	232.2	232.4	231.5	230.7
		SD	8.83	8.17	7.75	9.33
		N	12	12	12	12
		% of control	-	100	100	99
Treatment Period	Day 1 (Wi)	Mean	237.1	237.6	235.6	235.2
		SD	12.22	9.52	9.69	10.95
		N	12	12	12	12
		% of control	-	100	99	99
	Day 4 (Wi)	Mean	236.4	239.3	237.8	235.2
		SD	11.89	7.04	13.00	11.85
		N	12	12	12	12
		% of control	-	101	101	99
	Day 8 (Wi)	Mean	239.6	240.7	239.0	237.1
		SD	11.28	8.12	12.85	12.44
		N	12	12	12	12
		% of control	-	100	100	99
	Day 11 (Wi)	Mean	235.8	239.4	238.3	235.8
		SD	10.65	7.98	12.20	12.51
		N	12	12	12	12
		% of control	-	101	101	100
	Day 14 (Wi)	Mean	238.2	239.2	237.5	237.1
		SD	11.52	7.23	10.32	12.16
		N	12	12	12	12
		% of control	-	100	100	100
Gestation Period	Day 0 (Wi)	Mean	240.7	243.4	241.3	239.2
		SD	12.20	8.49	10.58	12.48
		N	12	12	12	12
		% of control	-	101	100	99
	Day 4 (Wi)	Mean	255.3	255.5	255.5	253.1
		SD	11.05	9.38	13.72	12.71
		N	12	12	12	12
		% of control	-	100	100	99
	Day 7 (Wi)	Mean	263.0	264.3	263.3	261.6
		SD	12.05	10.18	14.80	14.02
		N	12	12	12	12
		% of control	-	101	100	99
	Day 11 (Wi)	Mean	277.4	278.4	275.6	275.4
		SD	12.96	10.73	15.85	15.05
		N	12	12	12	12
		% of control	-	100	99	99
	Day 14 (Wi)	Mean	286.8	288.9	287.9	289.5
		SD	17.91	11.15	16.76	15.64
		N	12	12	12	12
		% of control	-	101	100	101
	Day 17 (Wi)	Mean	305.6	312.8	310.3	313.5
		SD	23.87	11.48	17.4	19.21
		N	12	12	12	12
		% of control	-	102	102	103
	Day 20 (Wi)	Mean	328.0	339.9	339.7	338.2
		SD	29.46	15.08	18.70	23.98
		N	12	12	12	12
		% of control	-	104	104	103
Lactation Period	Day 1 (Wi)	Mean	253.5	249.0	250.2	254.7
		SD	15.27	15.58	15.94	17.73
		N	10	12	12	11
		% of control	-	98	99	100
	Day 4 (Wi)	Mean	269.2	268.9	270.6	276.8
		SD	10.74	14.68	14.62	16.43
		N	9	12	11	11
		% of control	-	100	101	103
	Day 7 (Wi)	Mean	278.8	275.4	277.4	283.3
		SD	8.11	15.07	12.72	18.41
		N	9	12	11	11
		% of control	-	99	100	102
	Day 14 (Wi)	Mean	288.8	295.4	294.4	297.0
		SD	12.62	18.49	18.41	22.64
		N	9	12	11	11
		% of control	-	102	102	103
	Day 17 (Wi)	Mean	296.3	293.8	297.4	295.4
		SD	15.36	14.64	16.52	22.23
		N	9	12	11	11
		% of control	-	99	100	100

Table 10. F0 FOB: Motor Activity - group meanabsolute values (beam counts)
Dose Group	Motor Activity	5 (min)	10 (min)	15 (min)	20 (min)	25 (min)	30 (min)	Total (min)
Males
Statistics Test		Wi	Wi	Wi	Wi	Wi	Wi	Wi
Control	Mean	179.4	138.4	86.2	126.0	82.8	46.8	659.6
	SD	30.36	58.54	58.26	53.83	48.85	43.07	139.68
	N	5	5	5	5	5	5	5
150 mg/Kg/day	Mean	201.8	76.2	41.0	109.4	69.4	22.2	520.0
	SD	100.08	75.41	26.33	74.33	75.93	16.35	260.34
	N	5	5	5	5	5	5	5
	% of control	112	55	48	87	84	47	79
300 mg/Kg/day	Mean	199.4	141.2	124.8	95.4	64.6	101.2	726.6
	SD	70.22	77.39	85.46	60.39	58.05	72.79	220.35
	N	5	5	5	5	5	5	5
	% of control	111	102	145	76	78	216	110
600 mg/Kg/day	Mean	276.4	199.4	134.8	136.6	88.2	18.8	854.2
	SD	126.82	77.68	51.69	54.48	65.72	21.79	288.94
	N	5	5	5	5	5	5	5
	% of control	154	144	156	108	107	40	130
Females
Control	Mean	160.2	78.8	73.8	44.0	53.0	48.8	458.6
	SD	19.32	50.26	57.80	46.82	47.75	80.42	205.16
	N	5	5	5	5	5	5	5
150 mg/Kg/day	Mean	111.6	69.2	22.4	39.8	44.8	27.6	315.4
	SD	80.62	55.93	32.35	38.00	39.47	40.83	195.63
	N	5	5	5	5	5	5	5
	% of control	70	88	30	90	85	57	69
300 mg/Kg/day	Mean	147.4	124.2	33.4	8.8	22.6	26.2	362.6
	SD	26.23	97.49	58.06	8.61	29.11	43.38	154.14
	N	5	5	5	5	5	5	5
	% of control	92	158	45	20	43	54	79
600 mg/Kg/day	Mean	262.8**	152.2	116.2	55.4	24.0	25.0	635.6
	SD	37.25	43.96	62.35	43.66	38.52	30.28	174.61
	N	5	5	5	5	5	5	5
	% of control	164	193	157	126	45	51	139

* p≤0.05

** p≤0.01

Table 11. F0 FOB: Grip Strength - group mean absolute values (g)
Dose Group		Forelimb Mean (g)	Hindlimb Mean (g)
Statistics Test		Wi	Wi
Males
Control	Mean	880.01	777.97
	SD	104.052	175.758
	N	5	5
150 mg/Kg/day	Mean	930.38	891.20
	SD	104.071	230.406
	N	5	5
	% of control	106	115
300 mg/Kg/day	Mean	914.08	901.83
	SD	127.802	104.196
	N	5	5
	% of control	104	116
600 mg/Kg/day	Mean	897.09	877.35
	SD	72.559	165.634
	N	5	5
	% of control	102	113
Females
Control	Mean	860.65	765.07
	SD	34.236	53.565
	N	5	5
150 mg/Kg/day	Mean	896.43	760.92
	SD	34.236	25.793
	N	5	5
	% of control	104	99
300 mg/Kg/day	Mean	957.66*	724.79
	SD	58.709	82.191
	N	5	5
	% of control	111	95
600 mg/Kg/day	Mean	940.01*	677.67*
	SD	53.845	22.658
	N	5	5
	% of control	109	89

Wi: Treated groups compared with Control using Williams’ test

* p≤0.05

** p≤0.01

Table 12. F0 Hematology and Coagulation - Group mean value
Dose Group		Neutrophils (x 10^9/L)	Basophils (x 10^9/L)	LRT (x 10^12/L)
Statistics Test		Wi	Wi	Wi
Males
Control	Mean	0.54	0.01	0.097
	SD	0.148	0.004	0.0179
	N	5	5	5
150 mg/Kg/day	Mean	0.49	0.01	0.088
	SD	0.085	0.004	0.0160
	N	5	5	5
300 mg/Kg/day	Mean	0.57	0.01	0.089
	SD	0.149	0.004	0.0133
	N	5	5	5
600 mg/Kg/day	Mean	0.88*	0.02**	0.090
	SD	0.373	0.011	0.0111
	N	5	5	5
Females
Control	Mean	0.089	0.01	0.0115
	SD	0.313	0.007	0.0133
	N	5	5	5
150 mg/Kg/day	Mean	0.82	0.01	0.106
	SD	0.125	0.004	0.0172
	N	5	5	5
	% of control	91	120	92
300 mg/Kg/day	Mean	0.66	0.01	0.113
	SD	0.078	0.000	0.0130
	N	5	5	5
	% of control	74	100	98
600 mg/Kg/day	Mean	1.27	0.02	0.093*
	SD	0.433	0.009	0.0159
	N	5	5	5
	% of control	142	160	93

Wi: Treated groups compared with Control using Williams’ test

* p≤0.05

** p≤0.01

LRT: Reticulocyte maturity index

Table 13. F0 Clinical Biochemistry - Group mean values post mating
Dose Group		Protein (g/L)	Globulin (g/L)
Statistics Test		Wi	Wi
Males
Control	Mean	62.14	22.48
	SD	1.80	1.70
	N	5	5
150 mg/Kg/day	Mean	63.84	24.66
	SD	2.02	2.06
	N	5	5
300 mg/Kg/day	Mean	65.08*	25.12*
	SD	2.64	2.59
	N	5	5
600 mg/Kg/day	Mean	68.82**	26.96**
	SD	2.13	1.09
	N	5	5
Females
Control	Mean	53.64	17.62
	SD	1.849	2.364
	N	5	5
150 mg/Kg/day	Mean	53.62	17.38
	SD	1.743	2.009
	N	5	5
	% of control	100	99
300 mg/Kg/day	Mean	55.66	17.72
	SD	1.716	1.571
	N	5	5
	% of control	104	101
600 mg/Kg/day	Mean	55.20	19.02
	SD	2.758	2.123
	N	5	5
	% of control	103	108

Wi: Treated groups compared with Control using Williams’ test

* p≤0.05

** p≤0.01

Table 14. F0 Organ weights - group mean absolute and adjusted values (g)
Dose Group		Organ
		Kidneys	Liver	Spleen	Thymus	Uterus and Cervix and Oviducts
Statistics Test		Wi	Wi	Wi	Wi	Sh
Males
Control	Mean	2.237	10.908	0.602	0.370	-
	SD	0.194	0.866	0.027	0.044	-
	N	5	5	5	5	-
	Adjusted Mean	2.252	11.137	0.621	0.378	-
150 mg/Kg/day	Mean	2.313	13.045	0.668	0.295	-
	SD	0.231	2.330	0.138	0.067	-
	N	5	5	5	5	-
	% of control	103	120	111	80	-
	Adjusted Mean	2.312	13.021	0.666	0.294	-
300 mg/Kg/day	Mean	2.431	12.628	0.626	0.278	-
	SD	0.172	2.633	0.114	0.078	-
	N	5	5	5	5	-
	% of control	109	116	104	75	-
	Adjusted Mean	2.416	12.387	0.605	0.270	-
600 mg/Kg/day	Mean	2.612	15.260	0.736	0.304	-
	SD	0.205	1.395	0.083	0.068	-
	N	5	5	5	5	-
	% of control	117	140	122	82	-
	Adjusted Mean	2.615*	15.296**	0.739*	0.306*	-
Females
Control	Mean	1.808	10.381	0.680	0.219	0.730
	SD	0.131	0.802	0.089	0.039	0.229
	N	5	5	5	5	9
	Adjusted Mean	1.810	10.394	0.681	0.219	0.731
150 mg/Kg/day	Mean	1.783	10.650	0.699	0.239	0.664
	SD	0.104	0.398	0.101	0.061	0.129
	N	5	5	5	5	12
	% of control	99	103	103	109	91
	Adjusted Mean	1.795	10.723	0.702	0.239	0.667
300 mg/Kg/day	Mean	1.931	10.859	0.705	0.232	0.618
	SD	0.074	0.496	0.095	0.040	0.117
	N	5	5	5	5	11
	% of control	107	105	104	106	85
	Adjusted Mean	1.943	10.927	0.708	0.232	0.619
600 mg/Kg/day	Mean	1.801	11.624	0.673	0.213	0.563
	SD	0.230	1.887	0.101	0.038	0.146
	N	5	5	5	5	11
	% of control	100	112	99	97	77
	Adjusted Mean	1.776	11.471	0.666	0.214	0.559*

Wi: Treated groups compared with Control using Williams’ test

Sh: Treated groups compared with Control using Shirley’s test

* p≤0.05

** p≤0.01

- Not applicable

Table 15. Mean plasma TSH concentrations (pg/mL)
Dose Group		F0 Males	F0 Female on Day 21 Post-partum
Statistics Test		Wi	Sh
Control	Mean	1560	1860
	SD	1010	N/A
	N	12	9
150 mg/Kg/day	Mean	1800	543
	SD	749	134
	N	12	12
300 mg/Kg/day	Mean	1550	607
	SD	685	20
	N	10	11
600 mg/Kg/day	Mean	1350	751
	SD	571	472
	N	10	10

Wi: Treated groups compared with Control using Williams’ test

Sh: Treated groups compared with Control using Shirley’s test

* p≤0.05

** p≤0.01

 

Table 16. Mean serum T4 concentrations (pg/mL)
Dose Group		F0 Males	F0 Female on Day 21 Post-partum
Statistics Test		Wi	Sh
Control	Mean	47600	39578
	SD	6512	5434
	N	12	9
150 mg/Kg/day	Mean	49158	39333
	SD	6331	6447
	N	12	12
300 mg/Kg/day	Mean	44650	37209
	SD	5207	3529
	N	10	11
600 mg/Kg/day	Mean	40109**	39191
	SD	3739	7049
	N	11	11

Wi: Treated groups compared with Control using Williams’ test

Sh: Treated groups compared with Control using Shirley’s test

* p≤0.05

** p≤0.01

Table 17. Summary of Reproductive Performance
Treatment Group	Control	150 mg/Kg/day	300 mg/Kg/day	600 mg/Kg/day
Total Females	12	12	12	12
Unscheduled Deaths Prior to Pairing	0	0	0	0
Females Paired	12	12	12	12
Unscheduled Deaths Prior to Pairing	0	0	0	0
Females Mated	12	12	12	12
Pregnant Females	12	12	12	12
Euthanized for welfare reasons	1	0	0	1
Total litter loss	1	0	1	0
Failed to litter	1	0	0	0
Non Pregnant Females	0	0	0	0
Not Parturition	2	0	0	0
Number of Conception Day
Day 1	3	0	2	1
Day 2	4	3	2	2
Day 3	5	6	6	6
Day 4	0	3	2	3
Percentage Mating (%)	100	100	100	100
Conception rate (%)	100	100	100	100
Fertility Index (%)	100	100	100	100
Gestation Index (%)	75	100	100	100
Post-Implantation Loss (%)	12.2	7.8	6.2	9.1
Estrous cycle (mean length)	4.0	4.0	4.0	4.2
Females showing evidence of copulation	12	12	12	12
Females achieving pregnancy	12	12	12	12
Dams with live young
Day 1	9	12	12	12
Day 4	9	12	11	11
Implants / dam (mean)	11.7	13.8	12.3	13.3
Live pups / dam
Day 1 (mean)	9.9	11.9	11.6	10.4
Day 4 (mean)	9.6	11.8	11.2	10.0
Sex Ration (M:F)
Day 1 (mean)	0.98	0.86	0.99	0.79
Day 4 (mean)	0.95	0.88	0.98	0.77
Litter weight
Day 1 (mean)	5.64	5.31	5.49	5.62
Day 4 (mean)	8.15	7.85	8.08	9.00
Day 13 (mean)	30.17	29.93	28.66	31.29
Pup weight: Males
Day 1 (mean)	5.82	5.40	5.61	5.75
Day 4 (mean)	8.36	7.92	8.21	9.14
Day 13 (mean)	30.53	30.08	28.98	31.53
Pup weight: Females
Day 1 (mean)	5.47	5.22	5.36	5.49
Day 4 (mean)	7.94	7.78	7.95	8.85
Day 13 (mean)	29.81	29.77	28.35	31.05
Pup AGD Males Day 4 (mean)	3.54	3.39	3.28	3.50
Pup AGD Females Day 4 (mean)	1.39	1.50	1.47	1.55
Loss of offspring
Pre-natal / post-implantations (implantation minus live births)
Females with 0	3	2	6	3
Females with 1	0	3	3	2
Females with 2	3	5	3	1
Females with ≥3	3	2	0	6
Post-natal (live births minus alive on Day 4)
Females with 0	2	2	1	2
Females with 1	2	0	0	0
Females with 2	1	0	1	2
Females with ≥3	4	10	10	8

Table 18. F1 Food consumption - group mean values (g/animal/day)
Dose Group		Days 1-8	Days 8-15	Days 15-22	Days 22-29	Days 29-36	Days 36-43	Days 43-50
Statistical Test		Wi	Wi	Wi	Wi	Wi	Wi	Wi
Males
Control	Mean	9	15	19	19	20	20	20
	SD	0.8	3.5	1.8	1.4	2.0	1.6	1.1
	N	9	9	9	9	9	9	9
150 mg/Kg/day	Mean	8	15	19	19	20	20	19
	SD	0.8	4.4	1.6	1.1	1.4	1.5	1.4
	N	12	12	12	12	12	12	12
	% of Control	94	102	101	100	98	99	98
300 mg/Kg/day	Mean	8	15	20	20	21	21	20
	SD	1.4	3.0	2.6	1.9	1.7	1.5	1.6
	N	11	11	11	11	11	11	11
	% of Control	96	103	104	103	103	103	101
600 mg/Kg/day	Mean	7*	15	20	21	21	21	20
	SD	1.5	3.8	2.0	1.5	1.8	1.8	1.7
	N	11	11	11	11	11	11	11
	% of Control	86	102	103	106	103	104	102
Females
Control	Mean	8	13	15	14	15	14	13
	SD	0.9	1.6	1.1	1.3	2.7	1.2	0.9
	N	9	9	9	9	9	9	9
150 mg/Kg/day	Mean	8	13	15	14	14	15	14
	SD	0.6	1.2	1.2	0.8	1.8	2.0	1.0
	N	12	12	12	12	12	12	12
	% of Control	98	101	100	103	97	106	104
300 mg/Kg/day	Mean	8	14	15	15	15	15	14
	SD	1.1	1.8	1.4	2.2	3.2	1.5	1.3
	N	11	11	11	11	11	11	11
	% of Control	98	106	104	108	100	104	107
600 mg/Kg/day	Mean	7	13	16	15	14	15	14
	SD	1.4	2.4	1.3	1.1	1.4	1.2	1.1
	N	11	11	11	11	11	11	11
	% of Control	92	103	107	108	97	104	106

Table 19. Litter Responses - F0/F1 Generations - group mean values
Dose Group		Live birth index (%)	Viability index Day 4 (%)	Viability index Day 13 (%)	Viability index Day 21 (%)	Postnatal loss Day 4 (%)	Postnatal loss Day 13 (%)	Postnatal loss Day 21 (%)
Statistics Test		Fe	Fe
Females
Control	Mean	84.5	91.9	100.0	100.0	8.1	0.0	0.0
	SD	31.29	14.03	0.00	0.00	14.03	0.00	0.00
	N	10	9	9	9	9	9	9
150 mg/Kg/day	Mean	94.3	98.8	100.0	100.0	1.2	0.0	0.0
	SD	8.65	2.80	0.00	0.00	2.80	0.00	0.00
	N	12	12	12	12	12	12	12
	% of Control	112	107	100	100	15	100	100
300 mg/Kg/day	Mean	100.0*	89.9	100.0	100.0	10.1	0.0	0.0
	SD	0.00	28.97	0.00	0.00	28.97	0.00	0.00
	N	12	12	11	11	12	11	11
	% of Control	118	98	100	100	125	100	100
600 mg/Kg/day	Mean	94.2	94.3	100.0	100.0	5.7	0.0	0.0
	SD	14.48	13.83	0.00	0.00	13.83	0.00	0.00
	N	12	11	11	11	11	11	11
	% of Control	112	103	100	100	71	100	100

Fe: Treated groups compared with Control using Fisher’s exact test

* p≤0.05

Table 20. F1 Macropathology - group distribution of findings
Tissue/Organ and Findings	Number of animals affected
	Males				Females
	Control	150 mg/Kg/day	300 mg/Kg/day	600 mg/Kg/day	Control	150 mg/Kg/day	300 mg/Kg/day	600 mg/Kg/day
Number of Animals	24	36	32	30	25	36	34	30
Number of animals within normal limits	23	36	32	30	23	34	33	30
Thymus Abnormal color	1	0	0	0	2	2	1	0

Table 21. F1 Organ weights - group mean absolute and adjusted values (g)
Dose Group		Terminal Body weight (g)	Thyroids and Parathyroids (g)
Males
Statistics Test		Wi	Sh
Control	Mean	292.2	0.017
	SD	20.1	0.005
	N	25	25
150 mg/Kg/day	Mean	286.5	0.014
	SD	27.6	0.003
	N	36	36
	% of Control	98	84
300 mg/Kg/day	Mean	292.0	0.016
	SD	26.1	0.004
	N	32	32
	% of Control	100	95
600 mg/Kg/day	Mean	288.8	0.015
	SD	24.0	0.003
	N	30	30
	% of Control	99	90
Females
Statistics Test		Wi
Control	Mean	194.3	0.014
	SD	13.8	0.004
	N	25	25
150 mg/Kg/day	Mean	192.2	0.012
	SD	13.7	0.003
	N	36	36
	% of Control	99	86
300 mg/Kg/day	Mean	195.0	0.014
	SD	14.0	0.004
	N	33	33
	% of Control	100	100
600 mg/Kg/day	Mean	198.3	0.013
	SD	17.4	0.004
	N	30	30
	% of Control	102	93

Applicant's summary and conclusion

Conclusions:

Based on the results observed, the systemic toxicity No Observed Adverse Effect Level (NOAEL) in males and females rats was determined to be 600 mg/Kg/day. At this dose level, histological findings (minimal to slight microscopic changes in the thyroid glands) observed were considered non-adverse in the absence of consistent changes in hormone levels. At 600 mg/Kg/day, thyroid hormone changes were observed. However, given that the mechanism leading to these changes is unknown, and in the absence of effects on reproductive performance, their relevance to humans is uncertain and could be therefore, considered non-adverse.
 
Based on lack of adverse treatment-related effects observed, the NOAEL for reproductive / developmental toxicity was determined to be 600 mg/Kg/day.

Executive summary:

A key Guideline 422 study was conducted to evaluate the effects of repeated oral (gavage) administration of the test material (P1400: 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2 (phenylmethyl) ester, CAS 1200806-67-2) to rats during a four-week period in F0 males two weeks before mating through a minimum of 28 days (after confirming female pregnancy), until day 20 postpartum for females including the day before sacrifice, and from birth to sexual maturation (PND 70) in F1 pups. 12 rats per sex per dose received the test material in corn oil once daily via oral gavage at does of 150, 300, or 600 mg/Kg/day while the control animals received corn oil only.

 

Animals were observed twice daily for mortality and for any clinical signs following administration of the test material. Parameters such as body weight, food consumption, behavioural assessments such as grip strength, motor activity, and sensory reactivity assessment were evaluated for. Additionally, blood samples were extracted from the retro-orbital plexus of all animals under light isoflurane anesthesia and clinical biochemistry as well as haematology assessments were undertaken. Blood samples were taken from all surviving adult males, at termination and from all surviving dams on day 21 postpartum for thyroid hormone analysis. At necropsy, post-mortem examination of the external surface of the body, all orifices, cranial, thoracic and abdominal cavities and their contents with emphasis on the uterus (females only), number of implantation sites (females only) was performed. Organ weights were recorded and all organ and tissue samples (except for the nose) of selected animals from the control and high dose groups (5 animals/sex) were processed, embedded, cut at an approximate thickness of 2-4 micrometers and stained with hematoxylin and eosin. Reproductive organs of all control and high dose animals as well as of animals selected for sexual maturation were also examined. Histopathological analysis was undertaken and slides of organs and tissues collected at necropsies of all animals to be evaluated, except for the bone marrow smear for which no test was planned, were examined by the pathologist. Additionally, based on treatment-related morphological changes detected in thyroids in high-dose animals, this organ from the other treatment group animals were examined in all adult animals. A qualitative staging of spermatogenesis and histopathology evaluation of interstitial cells of all males from the control and high-dose groups was also performed.

 

No treatment-related mortality, clinical signs (other than salivation) or effects on functional observation battery were observed in the parental generation. There was no effect on body weights during the study and there was no indication of an effect on food consumption during the treatment period. The statistically significant increases noted at 300 and 600 mg/Kg/day in males (postnatal days 8 and 15) or in females at 600 mg/Kg/day on gestation day 14 were not considered to have been toxicologically relevant given their magnitude (no more than 1.2 times the Control mean values), as it was transient and as there were no effects in body weights that could be associated with it. Haematology, coagulation and clinical biochemistry effects observed, in the absence of a dose-effect relationship (neutrophil and basophils) or given that the differences were only observed in one sex (protein and globulin values in males), were not considered to be toxicologically relevant. Gross necropsy did not reveal any remarkable findings and all findings were considered to be incidental and unrelated to treatment with the test material. Reproductive parameters of pre-coital, fertility or gestation length or index were unaffected by treatment. There were no effects on survival, litter size, sex ratio and no adverse effect on offspring growth. No adverse effects were observed in the reproductive organs relating to alterations in the sexual maturation.

 

In parental males, a dose-related increase in mean kidney weights, correlating to an increase in the amount of hyaline droplets, was observed at histopathological examination. However, this was considered to be within the normal historical range for rats of this strain and age. At 600 mg/Kg/day in parental animals, there were no histopathological findings that correlate with the increased liver and spleen weights in males or in the decrease in uterus, cervix and oviducts in females. A minimal or slight hypertrophy of the follicular epithelium of the thyroid glands was observed in males and females of all treated groups (150, 300 and 600 mg/Kg/day). This finding could be considered to be within the normal variation in females at 150 and 300 mg/Kg/day due to the low incidence recorded (2/12 at 150 mg/Kg/day and 1/11 at 300 mg/Kg/day), and the lack of effects in the T4 determinations. The decrease observed in T4 in parental males at 600 mg/Kg/day also correlated with the histopathological findings.

 

For the F1 generation, no changes in body weights or food consumption, clinical or necropsy signs were observed which were indicative of a reaction to the test material. There were no changes in the mean weights of the thyroids or parathyroids. In males, there was a dose-related increase in the incidence and/or severity of follicular cell hypertrophy in thyroid glands. The decrease in T4 and increase observed in TSH the PND 70 males at 600 mg/Kg/day also correlated with the histopathological findings. In females, minimal hypertrophy of the follicular epithelium in the thyroid glands was observed in a few animals treated at 600 mg/Kg/day (3 affected females from 30 observed).

 

Based on the results observed, the systemic toxicity No Observed Adverse Effect Level (NOAEL) in males and females rats was determined to be 600 mg/Kg/day. At this dose level, histological findings (minimal to slight microscopic changes in the thyroid glands) observed were considered non-adverse in the absence of consistent changes in hormone levels. At 600 mg/Kg/day, thyroid hormone changes were observed. However, given that the mechanism leading to these changes is unknown, and in the absence of effects on reproductive performance, their relevance to humans is uncertain and could be therefore, considered non-adverse.

 

Based on lack of adverse treatment-related effects observed, the NOAEL for reproductive / developmental toxicity was determined to be 600 mg/Kg/day.