Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-03-01 to 2017-06-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
extended one-generation reproductive toxicity - with both developmental neuro- and immunotoxicity (Cohorts 1A, 1B without extension, 2A, 2B, and 3)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-11-02 to 2017-07-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Deviations:
yes
Remarks:
Examinations of the F1-generation corresponded to OECD 408, recovery group included, prolonged duration (100d)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals:
Part 1 - Parental males (F0) were treated for 10 weeks pre-mating (this period fully covered the time required for epididymal transit of maturing spermatozoa and to allow the detection of post-testicular effects on sperm during the final stages of spermiogenesis and epididymal sperm maturation at mating). Parental females (F0) were treated for 10 weeks pre-mating (this period fully covered several complete oestrous cycles and was sufficient to detect any adverse effects on cyclicity).

- Basis for dose level selection: The dose levels (equivalent to dietary concentrations of 150, 500 and 1500 ppm) were selected by the Sponsor based on available data and information from previous experimental work, including the available results of a preliminary Dose Range Finding (DRF) palatability study (Study code: 15/094-220PE) and an OECD No. 422 study ((Study code: 15/094-220P) performed at the Test Facility.

In the OECD No. 422 study, OAPP was administered in diet at the concentrations of 300, 1250 and 5000 ppm, (equivalent to dietary doses of approximately 24.5, 97.1 and 337.6 mg/kg bw/day in the Low-, Mid- and High-dose groups, respectively) to Wistar rats for at least 28 days. Administration of OAPP caused significantly reduced body weights, reduced body-weight gain and reduced food consumption in the High dose of both sexes; slight differences were seen in the Mid-dose females. Organ weight and macroscopic observations at necropsy showed hepatic enlargement in both sexes at the High-dose and in Mid-dose males. At histopathology evaluation, hepatocellular vacuolation was also detected confirming the macroscopic observations. However, in the absence of effects on the liver function (as indicated by the clinical chemistry parameters) the hepatic changes were considered as probably an adaptive rather than an adverse effect. There was no effect of treatment on the examined reproductive parameters of male and female animals in the Low- and Mid-dose groups.However, test-item related adverse effects were seen in the reproductive parameters of females in the High-dose group as well as in the High-dose F1 generation (reduced viability, survival and development); these differences were considered to be probably secondary to maternal toxicity. The No Adverse Effect Level (NOAEL) of OAPP for systemic, reproductive and developmental toxicity was concluded as being 1250 ppm (97.1 mg/kg bw/day).

- Inclusion/exclusion of extension of Cohort 1B: Included (A sufficient number of pups (at least at least 20 animals/sex/dose group) were used for Cohort 1B.

- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: Included (at least 10 animals/sex/dose group were used for Cohorts 2A, 2B)

- Inclusion/exclusion of developmental immunotoxicity Cohort 3: Included (at least 10 animals/sex/dose group were used for Cohort 3. An additional 10 male and 10 female (age-matched) rats were used as satellite positive control animals for Cohort 3. (developmental immunotoxicity).

- Route of administration: oral (feed) - The oral route was selected, as it is a possible route of exposure to the test item in humans.

- Other considerations, e.g. on choice of species, strain, vehicle and number of animals:
Vehicle: corn oil used as the vehicle to facilitate mixing of the test item into the diet to obtain the desired dietary concentration.

Details on species / strain selection: The rat is regarded as suitable species for toxicology and reproduction studies. The Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. The same strain was used in the previous experimental work of the project performed at the Test Facility.

Number of animals:
Part 1: Parental (P) generation: 96 male and 96 female rats, 24 animals/sex/group, 4 groups. A sufficient number of spare animals was ordered; those animals were allocated to the spare colony of the Test Facility after the study had been finished.

Part 2: A sufficient number of pups (at least 20 animals/sex/dose group) of the F1 generation were used to perform a 90-day repeated dose toxicity study, including 30-day recovery groups (5 animals/sex in the Control and High dose group). This served as Cohort 1A (this cohort was used for the OECD 408 study) of the EOGRT study.

Other cohorts: A sufficient number of pups (at least at least 20 animals/sex/dose group for Cohort 1B (this cohort was used for the OECD 408 study), or at least 10 animals/sex/dose group for Cohorts 2A, 2B and 3) were used for additional cohorts of the EOGRT study.

Satellite animals: Additional 10 males and 10 females (age-matched) animals were used as satellite positive control animals for Cohort 3 (developmental immunotoxicity).
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: RÜTGERS Novares GmbH, batch No. 38900
- Composition of test material: composition is specified in IUCLID Sect. 13 - Assessment reports under Certificate of Analysis_Novares LA 300_phenol, methylstyrenated
- Expiration date of the lot/batch: 13 February 2018
- Purity test date:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, <70 RH%), protected from light
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:

FORM AS APPLIED IN THE TEST (if different from that of starting material): Light yellow to colourless liquid, at low temperature tendency to crystallisation

OTHER SPECIFICS:
- Purity: 100% (UVCB)
- Name of test material (as cited in study report): Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol, OAPP
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI
Details on species / strain selection:
The rat is regarded as suitable species for toxicology and reproduction studies. The Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. The same strain was used in the previous experimental work of the project performed at the Test Facility.
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Address: Sandhofer Weg 7, D-97633 Sulzfeld, Germany) from SPF colony
- Females (if applicable) nulliparous and non-pregnant: yes (Females were nulliparous and non-pregnant)
- Age at study initiation: Young adult rats, at least 6 weeks old at the initiation of study, and at least 16 weeks at mating.
- Weight at study initiation: Males: 244-285 g, Females: 171-214 g at the start of the treatment; values did not exceed ± 20% of the mean weight for each sex.
- Fasting period before study: No
- Housing: Parental animals were group-housed, up to 3 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or individually housed, respectively. Males were caged individually after mating had been completed. Animals of F1 generation were group-housed (2 animals of the same sex per cage) after the cohort selection, wherever possible. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by ssniff Spezialdiäten GmbH, (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany) ad libitum
- Water (e.g. ad libitum): tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY: Food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15.2 – 27.0°C (target range: 22 ± 3°C)
- Humidity (%): 20 – 74% (target range: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light (from 6.00 a.m. to 6.00 p.m.)

IN-LIFE DATES: From: To:
Route of administration:
oral: feed
Vehicle:
corn oil
Remarks:
used as the vehicle to facilitate mixing of the test item into the diet to obtain the desired dietary concentration.
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Each diet concentration was prepared as one batch of 100-120 kg/concentration level at the first preparation time, and variable amounts at the further preparation time (30-200 kg/concentration level), depending on the available stability data and/or the expiry date of the diet.
- Mixing appropriate amounts with (Type of food): The test material (OAPP) was incorporated into ssniff® SM R/M-Z+H “Complete Feed for Rats and Mice-maintenance” by ssniff Spezialdiäten GmbH (D-59494 Soest, Germany) to generate the test concentrations required for the study (150, 500 and 1500 ppm). Test material (OAPP) was incorporated into the diet using a solution in corn oil and mixed for up to approximately 12 minutes (approximately 6 minutes for premix preparation, and 6 minutes for preparation of the complete diets). The diets were always produced in an ascending order regarding the test item concentration. Similar diet preparation procedures were used to generate control diet (0 mg OAPP/kg diet).
- Storage temperature of food: Prepared diets were stored at room temperature under dry conditions in sewed bags pending and during transport to CiToxLAB Hungary Ltd. At CiToxLAB Hungary Ltd., the prepared diet bags were stored in areas designated for diet storage at room temperature (approximately 15-21°C), under dry conditions, pending transfer to animal room at approximately 22 ± 3°C for animal feeding.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn Oil (Corn oil was used as the vehicle to facilitate mixing of the test item into the diet to obtain the desired dietary concentration) supplied by Henry Lamotte GmbH (Bremen, Germany)
- Concentration in vehicle: 4%
- Lot/batch no. (if required): Batch Numbers: 16-3329 (8001046002) / 16-3424 (8001213001), 16-3481 (8001409001) / 17-3551 (8002635001); Expiry dates: November 2017 / March 2018 / May /2018 / July 2018)
- Purity: A quality of Ph.Eur.8 (2014) & BP 2016
Details on mating procedure:
Part 1 animals
- M/F ratio per cage: 1:1 (Note: In one case, as a High-dose male (#4019) died during the pre-mating period, its pair female (#4519) was placed in a cage with another male and female of the same group (#4018 and #4518), as no individual male was available (2:1 mating).
- Length of cohabitation: Females remained with the same male until copulation occurred.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: Not specified
- After successful mating each pregnant female was caged: Sperm positive females were caged individually.
- Other: Part 2 animals

No mating was performed for Cohort 1A animals which were used to populate the OECD 408 study.

Other cohorts: No mating was performed for other cohorts of F1 generation (Cohort 1B, 2, and 3).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the diets for homogeneity and concentration of OAPP was performed at the designated Test Site (Test Site #1) using a validated GC method [6]. Test-item containing diet samples of an appropriate weight were collected from at least five different places of the diet container (in duplicates) from each dose group at least before the start, in the middle and near the end of the use of each batch of diets during the study to determine concentration and homogeneity. At least one sample (in duplicate) was taken from the control diet for concentration analysis at each occasion.
Duration of treatment / exposure:
Part 1 animals
Parental males (F0) were treated for 10 weeks pre-mating (this period fully covered the time required for epididymal transit of maturing spermatozoa and to allow the detection of post-testicular effects on sperm during the final stages of spermiogenesis and epididymal sperm maturation at mating), through the mating period and after that until they were no longer needed for assessment of reproductive effects (i.e. after the placental sign evaluation had been finished for all mated adult females, by the time the deliveries had been also started); a total of at least 13 weeks of treatment.

Parental females (F0) were treated for 10 weeks pre-mating (this period fully covered several complete oestrous cycles and was sufficient to detect any adverse effects on cyclicity), and during the mating, gestation, and lactation periods until they were no longer needed for assessment of reproductive effects (i.e. after weaning of their litters; on PPD 23); a total of at least 16 weeks of treatment.

Part 2 animals
Male and female pups born in Part 1 were used for Part 2 (Cohort 1A - used for the OECD 408 study) of the study. After weaning (on PND 21), animals were treated for 100 days. Recovery animals (Control and High-dose groups) of this cohort were kept for 30 additional days after the treatment had finished and during this period they were fed the study control diet.

Other cohorts
Animals of other cohorts were treated the same way as Part 2 (Cohort 1A) animals.
Frequency of treatment:
Rats fed a constant concentration of OAPP in diet (ad libitum)
Details on study schedule:
No mating was performed for Cohort 1A animals (used to populate the OECD 408 study). No mating was performed for other cohorts of F1 generation (including Cohort 1B - used for the OECD 408 study).
Dose / conc.:
0 ppm
Remarks:
Control
Dose / conc.:
150 ppm (nominal)
Remarks:
Low Concentration (150 mg/kg diet); corresponds to 12 / 13 mg/kg bw/day (actual dose received) // (P0 / F1 generation, measured, see Tab. 10 below)
Dose / conc.:
500 ppm (nominal)
Remarks:
Intermediate Concentration (500 mg/kg diet); corresponds to 40 / 42 mg/kg bw/day (actual dose received) // (P0 / F1 generation, measured, see Tab. 10 below)
Dose / conc.:
1 500 ppm (nominal)
Remarks:
High Concentration (1500 mg/kg diet); corresponds to 124 / 130 mg/kg bw/day (actual dose received) // (P0 / F1 generation, measured, see Tab. 10 below)
No. of animals per sex per dose:
Part 1: Parental (P) generation: 24 animals/sex/concentration
Part 2: F1 generation: Cohort 1A: at least 20 animals/sex/concentration (used to populate the OECD 408 study); Cohort 1B: at least 20 animals/sex/concentration (used to populate the OECD 408 study); Cohorts 2A, 2B, and 3: at least 10 animals/sex/concentration
Satellite animals: Additional 10 males and 10 females (age-matched) animals were used as satellite positive control animals for Cohort 3 (developmental immunotoxicity).
Control animals:
yes, concurrent vehicle
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels (equivalent to dietary concentrations of 150, 500 and 1500 ppm) were selected by the Sponsor based on available data and information from previous experimental work, including the available results of a preliminary Dose Range Finding (DRF) palatability study (Study code: 15/094-220PE) and an OECD No. 422 study ((Study code: 15/094-220P) performed at the Test Facility.

In the OECD No. 422 study, OAPP was administered in diet at the concentrations of 300, 1250 and 5000 ppm, (equivalent to dietary doses of approximately 24.5, 97.1 and 337.6 mg/kg bw/day in the Low-, Mid- and High-dose groups, respectively) to Wistar rats for at least 28 days. Administration of OAPP caused significantly reduced body weights, reduced body-weight gain and reduced food consumption in the High-dose of both sexes; slight differences were seen in the Mid-dose females. Organ weight and macroscopic observations at necropsy showed hepatic enlargement in both sexes at the High-dose and in Mid-dose males. At histopathology evaluation, hepatocellular vacuolation was also detected confirming the macroscopic observations. However, in the absence of effects on the liver function (as indicated by the clinical chemistry parameters) the hepatic changes were considered as probably an adaptive rather than an adverse effect. There was no effect of treatment on the examined reproductive parameters of male and female animals in the Low- and Mid-dose groups.However, test-item related adverse effects were seen in the reproductive parameters of females in the High-dose group as well as in the High-dose F1 generation (reduced viability, survival and development); these differences were considered to be probably secondary to maternal toxicity. The No Adverse Effect Level (NOAEL) of OAPP for systemic, reproductive and developmental toxicity was concluded as being 1250 ppm (97.1 mg/kg bw/day).

- Rationale for animal assignment (if not random): All parental (P) animals were sorted according to body weight by computer and divided into weight ranges. There were an equal number of animals from each weight group in each of the experimental groups assigned by randomization according to the actual body weight to ensure that animals of all test groups were as close as practicable to a uniform weight. This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the group.

Males and females were randomised separately.
Positive control:
An immunosuppressive positive control substance (cyclophosphamide monohydrate) was administered to animals assigned to positive control satellite group (Group 9).
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Adult animals inspected for signs of morbidity and mortality twice daily, at the beginning and end of the working day as practical. General clinical observations were made once a day (no general clinical observations were made on those days when detailed clinical observations were made). The animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including their onset, degree and duration as applicable.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all adult animals at least prior to the first treatment (to allow for within-subject comparisons) and weekly thereafter. These observations were made outside the home cage in a standard arena, at similar times, generally during the morning. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards). Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

The sperm positive females of Part 1 were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat) on Gestation Day 13 and/or 14 (GD13 / GD14).

Furthermore, mated females of Part 1 were examined carefully around the time of expected delivery for any signs of difficult or prolonged parturition.

BODY WEIGHT: Yes
- Time schedule for examinations:

Part 1 animals: Parental (P) males were weighed with a precision of 1 g at randomisation (Day -6), on the first day of treatment (Day 1), then at least weekly through the pre-mating / mating periods and after that, and prior to necropsy. Parental (P) females were weighed with the same precision at randomisation (Day -6), on the first day of treatment (Day 1), then at least weekly through the pre-mating / mating periods, on gestation days (GD) 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, and during lactation on PPD 0, 4, 7, 10, 14, 17 and 21 and prior to necropsy (or at death for animals found dead).

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

Food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g twice per week. Weekly food consumption was calculated for reporting purposes.

Part 1 animals: Food consumption of parental (F0) males and females was measured twice per week during the pre-mating period. Individual food consumption was not measured during cohabitation. Evaluation of food consumption of parental (F0) males commenced again following the mating period until necropsy and was conducted twice per week. Food consumption of mated F0 females was evaluated at least on days of body weight measurement (at least twice per week).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OTHER:

- Neurological assessment (Functional Observation Battery and SMART): Part 1 animals: No neurological assessment was performed for Part 1 adult animals (parental generation).

- Ophthalmology evaluation: Part 1 animals: No ophthalmoscopic examination was conducted for Part 1 adult animals (parental generation).

- Observation of the delivery process and nursing instinct

Part 1 animals: Pregnant parental (P) females were allowed to litter and rear their offspring. The delivery process was observed as carefully as possible (all observations were recorded). Furthermore, any evidence of abnormal deliveries was recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy (Gestational Day 0, GD0) until the completion of parturition.

Dams were observed for signs of nest building with the bedding material and for covering their new-borns. Evidence of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded.

For terminal blood sampling, 10 males and 10 females/group were selected. All animals selected for blood sampling were fasted (overnight period of food deprivation; in case of females this step was performed after the litter had been culled).

Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy. Four samples were taken from each animal: two tubes for haematology and thyroid hormone analysis (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.

Urine samples were collected overnight (for approximately 16 hours) with no access to food, but with access to water, in individual metabolism cages.

Haematology and blood clotting times

The parameters listed in Table# 4 were evaluated in selected Part 1 animals.

Clinical chemistry

The parameters listed in Table# 5 were evaluated in selected Part 1 animals.

Urinalysis

The parameters listed in Table# 6 were evaluated in selected Part 1 animals using the urine collected from the metabolism cages.

Thyroid hormone analysis
For thyroid hormone analysis of selected parental (P) animals, blood samples (collected into tubes containing K3-EDTA as anticoagulant) were kept on ice from sampling until centrifugation (within 30 minutes of collection), then centrifuged rapidly (1600 g / approx. 3000 rpm, 10 minutes, 4ºC). The resulting plasma was divided in at least two aliquots (volume target was at least 125 µL for the first aliquot and at least 75 µL for the second aliquot, remaining material - if any -was kept as third (back-up) aliquot) and stored in an ultrafreezer (-80±10°C) until shipping for analysis.
Oestrous cyclicity (parental animals):
Part 1 animals: Parental (P) females were monitored daily for the stage of oestrous cycle by assessment of vaginal smears for two weeks before the start of mating, and during the mating period until evidence of mating was noted.
The process of vaginal smear preparation is described in the mating procedure Section. The stage of oestrus was evaluated using these smears by competent personnel.
Sperm parameters (parental animals):
Parameters examined in all F0 male parental generations:
Part 1 animals: At termination for all F0 males, after the organ-weight measurement of testis and epididymis, one of each organ was collected for sperm evaluation as described below. The paired organ was preserved for histopathological examination.

Enumeration of homogenisation-resistant testicular spermatids and cauda epididymal sperm reserves was performed. In addition, sperm from the cauda epididymis were collected for determination of motility and sperm morphology. For measurement of sperm morphology, at least 200 sperm per sample were evaluated from fixed, wet preparations and classified as either normal or abnormal (fusion, isolated heads, misshapen heads and/or tails). For enumeration of spermatids, caudal epididymal reserves and morphology, samples from Control and High dose males were evaluated.

Sperm motility was evaluated immediately after sacrifice. The percentage of progressively motile sperms was determined subjectively. The smears for morphology were prepared and stored for later analysis, and also the testes and epididymides for homogenisation-resistant sperm counts. Analyses on these parameters were done on the Control and High dose group only; examination of Low and Mid dose groups were not deemed necessary.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 5 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:

Each litter of the parental females was examined as soon as possible after delivery (post-natal day 0 - PND0: when the delivery finished) to establish the number and sex of pups, stillbirths, live births, and the presence of gross abnormalities (including cleft palate; subcutaneous haemorrhages; abnormal skin colour or texture; presence of umbilical cord; presence of dried secretions).

In addition, the first clinical examination of the neonates included a qualitative assessment of body temperature, state of activity and reaction to handling.

Evidence of suckling was evaluated as of PND0, based on the presence or lack of the milk in pup’s stomach.

On PND 0, the anogenital distance of each pup was measured.

All litters were checked daily for the number of viable and dead pups. Live pups were weighed with accuracy of 0.01 g at birth (PND 0) and on PND 4, 7, 14 and 21.  

Clinical examinations, as applicable for the age of the animals, was repeated when the offspring were weighed, or more often if animal-specific findings had been made at birth. Signs noted included, but might not be limited to, external abnormalities, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity. Changes in gait, posture, response to handling, as well as the presence of clonic or tonic movements, stereotypy or bizarre behaviour were also recorded.

On PND 12 - 13, male pups were examined for retention of nipples/areolae.

On PND 21, at least three males and three females from each litter were chosen at random for continuation of treatment. The F1 pups selected for continuation were treated daily by the dietary route from weaning. All animals were examined once daily for mortality and morbidity. Clinical observations (for checking behavioural changes and any overt signs of toxicity) was made daily and a detailed clinical observation (thorough physical examination) was conducted weekly on each animal. The body weight of each pup was recorded at least weekly, commencing from PND 21. Food consumption was evaluated twice per week.

Females: Vaginal patency was evaluated daily for each F1 female (selected for cohorts) beginning PND 22 and any abnormality noted. The body weight of each female was recorded on the day of vaginal patency. Oestrous cycle was monitored for two weeks following vaginal patency; care was taken to avoid induction of pseudopregnancy.

Males: All F1 males (selected for cohorts) was evaluated for balanopreputial separation daily beginning on PND 35 to detect if sexual maturation occurs early. The body weight of each male was recorded upon separation.

BODY WEIGHT:
- Time schedule for examinations: Part 2 animals: Body weight was recorded with a precision of 1 g at weaning (PND 21, prior to the start of treatment which was designated as Day 1 of individual treatment), then at least weekly, including on Day 100 (last treatment day) and prior to necropsy (if scheduled necropsy, fasted, on Day 101).

Other cohorts: Body weight was recorded with a precision of 1 g at weaning (PND 21), then at least weekly and prior to necropsy (or at death for animals found dead).

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

Part 2 animals: The determination of food consumption of Cohort 1A (this cohort was used for the OECD 408 study) of F1 generation was performed twice per week. The first measurement of given food was made at weaning (PND 21, i.e. Day 1) The remaining, non-consumed food was weighed twice per week with a precision of 1 g. Food hoppers was replenished more frequently if required.

Other cohorts: The determination of food consumption was performed twice per week for other cohorts of F1 generation.

NEUROLOGICAL ASSESSMENT (Functional Observation Battery and SMART):

Part 2 animals: Towards the end of the treatment period, during Week 12/13, but not earlier than in Week 11, each animal of Cohort 1A (this cohort was used for the OECD 408 study) was subjected to the functional observation battery, including measurements of the landing foot splay and fore/hind limb grip strength; and motor activity assessment. A detailed assessment for neurotoxicity effects was made on the basis of these measurements.

Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed (Irwin, S., 1968).

Other cohorts: Similar neurological assessment was performed for Cohort 2A animals (young adults).
In case of evaluation of this cohort, the data from all groups were evaluated for travelled distance (in 5-minute segments). Graphical presentation (as in case of Cohort 1A animals) was also prepared for this cohort and used for comparison.

No examination was performed for other cohorts of F1 generation.

OPHTHALMOLOGY EVALUATION:

Part 2 animals: Ophthalmoscopic examination was conducted in all animals of Cohort 1A (this cohort was used for the OECD 408 study) soon after the start of treatment (Week 6) and in the Control (Group 5) and High dose
(Group 8) animals, during Week 12/13 of the treatment. As no treatment related alterations were found, no additional animals of Low (Group 6) and Mid (Group 7) dose groups or recovery animals were examined near/at termination.

Mydriasis was produced after instillation of “Humapent 5 mg/mL” (0.5% cyclopentolate hydrochloride) eye drops into the conjunctival sac. The evaluation was performed by external examination and using a WelchAllynTM (ID: 11730) or Gowlland (ID: 3124) ophthalmoscope, as practical.

Other cohorts: No ophthalmoscopic examination was conducted for other cohorts of F1 generation.

OESTROUS CYCLICITY:
Part 2 animals: The oestrous cycle was monitored daily in Cohort 1A (this cohort was used for the OECD 408 study) females for two weeks after vaginal opening.

Other cohorts: The same procedure was followed for other cohorts (except of Cohort 2B) as for Cohort 1A.

CLINICAL PATHOLOGY:
Part 2 (Cohort 1A - this cohort was used for the OECD 408 study) animals were terminated on Day 101 of their treatment period. Prior to scheduled necropsy, clinical pathology examinations (haematology, coagulation, clinical biochemistry and urinalysis) were conducted in all surviving animals.
Urine samples were collected overnight (for approximately 16 hours) with no access to food, but with access to water, in individual metabolism cages.

All the procedures of Cohort 1A were followed for Cohort 1B (in order to provide additional samples if needed in case of equivocal results of Cohort 1A. Both Cohorts, 1A and 1B were used for the OECD 408 study). No clinical pathology examinations were performed for any other cohorts of F1 generation.

Haematology and blood clotting times
All the parameters measured for selected parental generation (Part 1) animals (Table# 4) were also measured for all Cohort 1A (this cohort was used for the OECD 408 study) animals. The same parameters were measured for Cohort 1B (this cohort was used for the OECD 408 study). No such measurements were conducted for other cohorts.

Clinical chemistry
All the parameters measured for selected parental generation (Part 1) animals (Table# 5) were also measured for all Cohort 1A (this cohort was used for the OECD 408 study) animals. Additionally, the following parameters: Bile acids and Triglycerides were evaluated for all Part 2 animals. The same parameters were measured for Cohort 1B (this cohort was used for the OECD 408 study). No such measurements were conducted for other cohorts.

Urinalysis
All the parameters measured for selected parental generation (Part 1) animals (Table# 6) were also measured for all Cohort 1A (this cohort was used for the OECD 408 study) animals.The same parameters were measured for Cohort 1B (this cohort was used for the OECD 408 study). No such measurements were conducted for other cohorts.

Thyroid hormone analysis
Blood sampling was made for thyroid hormone analysis (TSH, T4 and/or T3) of Cohort 1A animals. Blood sampling for thyroid hormone analysis was made for all Cohort 1B animals (in order to provide sample if any additional data needed in case of equivocal results of Cohort 1A. Both Cohorts, 1A and 1B were used for the OECD 408 study).

Blood sampling and ELISA (Cohort 3 animals only)
On the day of scheduled termination, prior to euthanasia, blood was collected from each animal by heart puncture under pentobarbital anaesthesia to obtain serum for ELISA measurements (up to 2.4 mL blood as practical, in tubes with no anticoagulant). Blood samples were kept on ice from sampling until centrifugation (within 30 minutes of collection), then centrifuged rapidly (approx. 3000 rpm, 10 minutes, 4ºC). The resulting serum was divided into five aliquots of 100 µL (in polypropylene tubes) and stored in an ultrafreezer (at -80±10ºC) until analysis.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities: Pups found dead on PND 0 or at a later time were examined for possible defects and cause of death. All culled (surplus) pups were subjected at least to necropsy with detailed macroscopic external and internal examination for any abnormalities.

At weaning (on PND 21) pups from all available litters of F1 generation (up to 22 pups/sex/group) were selected for further examinations and maintained until sexual maturation (unless earlier testing was required). Pups were selected randomly, with the exception that obvious runts (animals with a body weight more than two standard deviations below the mean pup weight of the respective litter) were not normally included, as they were unlikely to be representative of the treatment group.

On PND 21, the selected F1 pups were randomly assigned to one of three cohorts* of animals, as follows:

• Cohort 1 (1A and 1B) = Reproductive/developmental toxicity testing (these cohorts were used for the OECD 408 study)
• Cohort 2 (2A and 2B) = Developmental neurotoxicity testing
• Cohort 3 = Developmental immunotoxicity testing

Cohort 1: Reproductive/developmental toxicity testing (animals from Cohort 1 (1A and 1B) were used for the OECD 408 study)

This cohort was subdivided into two separate groups, 1A and 1B.

Cohort 1A: At least one male and one female/litter/group (at least 20/sex/group) were selected for primary assessment of effects on the reproductive system and for assessment of general toxicity.

Animals were treated for 100 days after weaning (started on PND 21) including a 30-day recovery period in the Control and High concentratio group animals (5/sex/concentration).

Vaginal smears were examined daily for all Cohort 1A females, after the onset of vaginal patency, until the first cornified smear was recorded, in order to determine the time interval between these two events. The stage of oestrous cycle was also checked on day of termination.

At termination (on Day 101, after a 100-day long treatment period*), all procedures described in parental generation were followed (i.e. clinical pathology – including serum thyroid level assessment, sperm parameters in males/oestrous cycle in females, macroscopic examination, organ weight and tissue preservation, histopathology).

*Note: Due to technical reason (difficulties in sample shipment of sensitive splenic lymphocyte samples due to a national holiday at the Test Facility and Test Site #2), the 90-day treatment period of Cohort 1A animals was extended by 10 days (this cohort was used for the OECD 408 study).

Additionally, for the investigation of possible pre- and post-natally induced immunotoxic effects, Cohort 1A animals were subjected to extra steps at termination including weighing of extra organs/tissues associated with the immune system and splenic lymphocyte subpopulation analysis.

Cohort 1B (this cohort was used for the OECD 408 study): At least one male and one female/litter/group (at least 21/sex/group) was selected with the priority for follow-up assessment of reproductive performance by mating F1 animals, when assessed. No such an assessment was deemed necessary based on the available results of the study at the time of decision making as agreed by the Sponsor and Study Director. These animals were also dedicated to obtain additional histopathology data in cases of suspected reproductive or endocrine toxicants, or when results from Subset 1A were equivocal. This was not necessary.

The following organs were weighed and corresponding tissues processed to the block stage:

• Vagina (not weighed)
• Uterus with cervix
• Ovaries
• Testes (at least one)
• Epididymides
• Seminal vesicles and coagulating glands
• Prostate
• Pituitary
• Identified target organs

As histopathology results from Cohort 1A were not equivocal and no reproductive or endocrine toxicity was suspected, no additional histopathology of Cohort 1B animals was conducted.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Cohort 2: Developmental neurotoxicity testing

This cohort was subdivided into two separate groups, as of 2A and 2B.

Cohort 2A: At least a total of 20 pups per group (at least 10 males and 10 females per group; at least one male or one female per litter) were assigned for neurobehavioral testing followed by neurohistopathology assessment as adults.

An auditory startle test was performed on PND 24 (±1 day). The day of testing was counterbalanced across treated and control groups.

The startle test was performed individually. It consisted of a sudden, short and intense acoustic stimulus produced by a generator and transmitted to the animal which was restrained in a chamber positioned in a soundproof box. The startle response was detected by a movement sensor (force transducer mounted beneath a platform) placed under the chamber (Bioseb, Chaville France). The movement signal was amplified and converted by an analog-to-digital converter into voltage changes corresponding to the platform deflections. Each session consisted of 50 trials. In the auditory startle test, the mean response amplitude on each block of trials (5 blocks of 10 trials) was determined, with test conditions optimized to produce intra-session habituation.

An additional measurement of startle response was performed in the period of PND 63-75 for Cohort 2A animals.

At an appropriate time between PND 63 and PND 75, animals were subjected to a functional observational battery (including a modified Irwin test (Irwin, S., 1968) and measurements of the landing foot splay and fore/hind grip strength) and an automated test of motor activity (startle response).

Cohort 2B: At least a total of 20 pups per group (at least 10 males and 10 females per group; at least one male or one female per litter) were assigned for neurohistopathology assessment at weaning (PND 21/22).

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Cohort 3: Developmental immunotoxicity testing

Cohort 3: At least a total of 20 pups / group (at least 10 males and 10 females per group; at least one per litter, where possible) were assigned for developmental immunotoxicity testing using a T-cell dependent antibody response assay (TDAR) [12] at PND 56 (±3 days). Spare pups (from the control group) and animals of the same strain at the same age (obtained from stock or from same animal supplier) were used as positive control (satellite) animals study.

The response was evaluated by determining the titer of SRBC-specific IgM antibody in the serum by Enzyme Linked Immunosorbent Assay (ELISA), at the peak of the response. According to the scientific literature (Ladics, G.S., 2007), responses in rats typically peak six days after intravenous immunization.

Immunization and sample preparation

All animals in this cohort were treated with a single intravenous injection (tail vein) of sheep red blood cells (SRBC, 2x10^8 cells/rat) between PND 53 and 59, consistent with current immunotoxicity testing procedures (Ladics, G.S., 2007). Animals were sacrificed 6 days later (i.e. sRBC, on Day 0, euthanasia on Day 6), serum was prepared and analysed in a commercial ELISA kit for anti-SRBC IgM levels (Life Diagnostics, Inc., PA, USA: catalogue Number: 4200-2).

For SRBC preparation, blood of healthy Merino sheep* was used. Sterile sheep blood was stored 2-8oC according to the manufacturer’s instruction. SRBCs were prepared fresh on the day of use at the Test Facility at a concentration of 4x10^8 cells/mL in sterile PBS, and administered at a dose level/volume of 0.5 mL SRBCs preparation/rat.

An immunosuppressive positive control substance was administered to animals assigned to positive control satellite group (Group 9). The positive control substance (cyclophosphamide monohydrate, abbreviated as CP in the raw data and report) was formulated in phosphate-buffered saline (PBS) at a concentration of 5 mg/mL, apportioned into separate (daily) aliquots and stored refrigerated (2-8°C) until needed. Animals of the positive control group received the positive control substance at a dose volume of 5 mL/kg/day (dose level: 25 mg/kg bw/day) by intraperitoneal injection on the day of SRBC treatment and for 5 consecutive days (a total of six days, i.e. from Day 0 to Day 5) prior to the scheduled blood sampling time point.

Necropsy with blood sampling for TDAR was performed for this cohort on PND 62 ± 3.
Postmortem examinations (parental animals):
SACRIFICE
Part 1 animals
- Male animals: All surviving parental (P) adult males were terminated (one day after the last treatment) when they were no longer needed for assessment of reproductive effects
- Maternal animals: All surviving parental (P) adult females were terminated (one day after the last treatment) when they were no longer needed for assessment of reproductive effects

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

Unscheduled deaths

Gross pathology was performed on every animal irrespective of the date of its death. Dead animals were necropsied as early as possible after discovery. When necropsy was not an immediate action (in case of some found dead pups) the carcass was refrigerated (approximately 4°C) until necropsy could be performed.

Scheduled termination

Part 1 animals
One day after the last treatment, sodium pentobarbital administered by intraperitoneal injection, followed by exsanguination was used for euthanasia. After exsanguination the external appearance was examined; cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.

For all F0 females a vaginal smear was prepared and examined to determine the stage of the oestrous cycle at sacrifice. The number of implantation sites and corpora lutea was recorded in all cohabited females as applicable. At termination for all F0 males, after the organ-weight measurement of testis and epididymis, one of each organ was collected for sperm evaluation.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [8] were prepared for microscopic examination and weighed, respectively.

Organ Weights: Part 1 animals

The organs listed in Table #7 were trimmed of fat and weighed in surviving animals (parental generation and Cohort 1A (this cohort was used for the OECD 408 study) of F1 generation) at termination. Paired organs were weighed together except of testes and epididymides, which were weighed individually and were summarised. In case of necessity, for example if any significant difference in size was noted between paired organs, the individual weight of each organ was recorded. Absolute organ weights were measured; relative organ weights to the body and brain weights (if applicable) were calculated and reported.

Histopathology

Part 1 animals

In addition to the organs listed for organ-weight measurement, samples of peripheral nerve, muscle, spinal cord, eye plus optic nerve, gastrointestinal tract, urinary bladder, lung, trachea (with thyroid and parathyroid attached), bone marrow, vas deferens (males), mammary gland (males and females) and vagina were retained for parental (F0) animals in suitable medium* for histopathological examination.

Detailed histological examination was performed as follows:

• retained organs in the Control (Group 1) and High dose (Group 4) groups,
• all macroscopic findings (abnormalities), except of minor order from all animals
• retained reproductive organs (testes, epididymides, prostate, seminal vesicles with coagulation gland for males, and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Low and Mid dose groups suspected of reduced fertility (e.g., males failed to conceive / sire and females failed to deliver offspring), or for which oestrus cyclicity or sperm number, motility, or morphology were affected.

Detailed testicular histopathological examination was conducted in order to identify treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cell, or sloughing of cells into the lumen. Examination of the epididymis included evaluation of the caput, corpus and cauda using longitudinal sectioning.

Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary as well as the epithelial capsule and ovarian stroma. For females (evaluated 10 females/dose group), a qualitative depletion of the primordial and small growing follicle as well as corpora lutea in post-lactational ovary was evaluated.
Postmortem examinations (offspring):
SACRIFICE: Part 2 animals
Cohort 1A animals (this cohort was used for the OECD 408 study) were terminated on Day 101 of their treatment period. All the procedures for Cohort 1A were followed for animals of Cohort 1B (this cohort was used for the OECD 408 study). Following collection of blood for TDAR evaluation, satellite animals (Group 9) were euthanized by exsanguination under anaesthesia, while Cohort 3 animals were sacrificed and a gross necropsy was conducted.

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

Unscheduled deaths
Gross pathology was performed on every animal irrespective of the date of its death. Dead animals (some pups of F1 generation) were necropsied as early as possible after discovery. When necropsy was not an immediate action (in case of some found dead pups) the carcass was refrigerated (approximately 4°C) until necropsy could be performed. Dead pups were examined externally for gross abnormalities. Dead (intact) pups were necropsied with macroscopic examination in order to identify the probable cause of death.

Scheduled termination: Part 2 animals
Necropsy and macroscopic examination was performed on all surviving animals of Cohort 1A (this cohort was used for the OECD 408 study) at the end of treatment period (on Day 101) after the sample collection for clinical pathology was made. The animals were euthanized by exsanguination under pentobarbital anaesthesia similarly to the parental generation.

Prior to necropsy, the oestrus cycle of all females of Cohort 1A (this cohort was used for the OECD 408 study) was determined by taking vaginal smears.

All the procedures for Cohort 1A were followed for animals of Cohort 1B (this cohort was used for the OECD 408 study). Following collection of blood for TDAR evaluation, satellite animals (Group 9) were euthanized by exsanguination under anaesthesia, while Cohort 3 animals were sacrificed and a gross necropsy was conducted.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [7, 8] were prepared for microscopic examination and weighed, respectively.

Organ Weights: Part 2 animals

The organs listed in Table #7 were trimmed of fat and weighed in surviving animals (parental generation and Cohort 1A (this cohort was used for the OECD 408 study) of F1 generation) at termination. Paired organs were weighed together except of testes and epididymides, which were weighed individually and were summarised. In case of necessity, for example if any significant difference in size was noted between paired organs, the individual weight of each organ was recorded. Absolute organ weights were measured; relative organ weights to the body and brain weights (if applicable) were calculated and reported.

Additionally, for the investigation of possible pre- and postnatally induced immunotoxic effects, at termination animals of Cohort 1A were subject to weighing of the lymph nodes associated with and distant from the route of exposure (in addition to the weight of the adrenal glands, the thymus and the spleen, already performed in all Cohort 1A animals).

Cohort 2A - At termination, the brain of all animals in this cohort were fixed in formalin after opening the brain cavity, but with the brain supported by the skull base. After fixation, the brain was removed and weighed with a precision of 0.01 g. Weighing was approximately 48 hours after necropsy to prevent physical damage of the soft tissue during weighing.

Tissue preservation: Animals of cohort 2A and 2B were perfusion-fixed (using heparinised saline (1 unit/mL) first, then followed by 10% neutral buffered formalin).

Cohort 2A: After perfusion the cranial vault was opened, skin and internal organs from the abdominal and thoracic cavity were removed. The thigh muscles were dissected on both legs to expose sciatic nerve and gastrocnemius muscle. The Achilles tendon was cut through and carefully dissected upwards to remove gastrocnemius muscle and attached sciatic nerve. The distal end of tibial nerve was identified, then sciatic, sural and tibial nerves were removed from the muscle. Organs of the perfused animals were stored in the fixative for 48 hours (the brain remained in the cranium for this period to ensure the soft tissue was not deformed during the full fixation procedure).

Cohort 3

Following collection of blood for TDAR evaluation, satellite animals (Group 9) were euthanized by exsanguination under anaesthesia, while Cohort 3 animals were sacrificed and a gross necropsy was conducted. The following tissues (see the list below) were preserved in neutral buffered 10% formalin for possible future histopathological evaluation:

• Femur with articular surface (and bone marrow)
• Gross lesions
• Lymph node (mesenteric)
• Lymph node (axillary)
• Peyer’s patch (GALT)
• Spleen
• Sternum (with marrow)
• Thymus

However, as results from the other cohorts did not indicate a possible immunotoxic effect, no histopathology evaluation was made for any Cohort 3 animals.

Splenic lymphocyte isolation and lymphocyte subtyping: Part 2 (Cohort 1A) animals only.

Analysis of splenic lymphocyte subpopulations in non-immunised (Cohort 1A(this cohort was used for the OECD 408 study)) animals was performed to determine any exposure-related shifts in the immunological steady-state distribution of “helper” (CD4+) or “cytotoxic” (CD8+) thymus-derived lymphocytes or natural killer (NK) cells (rapid responses to neoplastic cells and pathogens); when compared with concurrent and/or historical control ranges.

Histopathology: Part 2 animals

On completion of the macroscopic examination, the tissues and organs listed in Table# 8 were retained from all Cohort 1A animals (this cohort was used for the OECD 408 study) in suitable medium. Vas deferens was also preserved for at least 20 males/group of Cohort 1A. Full histopathology was performed in Group 5 (Control) and Group 8 (High dose) and any animals found dead or euthanized pre-terminally during the study. In addition, any organs or tissues with macroscopic abnormalities (except minor changes) were subjected to histological examination from all groups.

Cohort 2A

Neurohistopathology was performed for the High dose and Control animals of each sex after termination. Multiple sections were examined from the brain to allow examination of olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum. The eyes (retina and optic nerve) and samples of peripheral nerve, muscle and spinal cord were examined.

For Cohort 2A and 2B, the preserved CNS and PNS tissues of perfused animals assigned to the Control and High dose group were processed to slides as detailed and subjected to a histopathology examination. The Mid and Low dose groups were processed to blocks at the same time as the other groups (to avoid shrinkage artefacts associated with prolonged storage in fixative). Histopathology evaluation of any macroscopic lesion was also performed.

Morphometric (quantitative) evaluations were performed on Cohorts 2A and 2B at the designated Test Site (Test Site #2) on representative areas of the brain (homologous sections carefully selected based on reliable microscopic landmarks) and included linear and/or areal measurements of specific brain regions.
Statistics:
Data were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., and then tabulated using the Microsoft Office Word and/or Excel, or collected using the software PROVANTIS v.9, as appropriate. Group means and standard deviations were calculated from numerical data obtained in the study.
The statistical evaluation of appropriate data (marked as † in the list) was performed with the statistical program package of SAS 9.2 (when using Provantis).
In case of the SAS 9.2 software package (within the validated Provantis system) the following decision tree was applied automatically for statistical evaluation of continuous numeric data:
The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.

If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control.
 
Reproductive indices:
1) Male Mating Index (%): (Number of males with confirmed mating / Total number of males cohabited) x 100
2) Female Mating Index (%): (Number of sperm-positive females / Total number of females cohabited) x 100
3) Male Fertility Index (%): (Number of males impregnating a female / Total number of males cohabited) x 100
4) Female Fertility Index (%): (Number of pregnant females / Number of sperm-positive females) x 100
5) Gestation Index (%): (Number of females with live born pups / Number of pregnant females) x 100
Offspring viability indices:
1) Viability (Survival) Index (%): (Number of live pups (at designated time) / Number of pups born) x 100
2) Intrauterine mortality (%): (Number of implantations-Number of live borns / Number of implantations) x 100
3) Sex ratio % (females): (Number of pups examined – Number of male pups / Number of pups examined) x 100
4) Developmental landmark (%): (Number of pups examined – Number of pups with response) / (Number of pups examined) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Minor clinical signs, not related to the test item treatment were detected for some parental animals during the study:
Thin fur was recorded for two control males, however this fact had no connection with the test item administration.
Piloerection was detected for one Low-dose female on Days 100-102. Red discharge from right eye was recorded for one Mid-dose female (#3505) in the period of Days 22-38 and for one High-dose female in the period of Days 15-28. Thin fur was recorded for a Mid-dose animal. None of these signs were considered as treatment-related.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item-related mortality was observed during the study.
A High-dose male was found dead on Day 36 (without any former clinical signs). Based on the isolated incidence, this fact was considered as unrelated to the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A clear test item-related decrease was noted in the mean body weight and in the body-weight gain of the High-dose group of both sexes, more pronounced in males (-9% and -15% vs. -5% and -10% in females on PPD21). Decreases also occurred in the Mid- and Low-dose groups, but without clear trends and without statistical significance. Usually the Mid-group result was close to the Low-group result, indicating a lack of evidence for an adverse effect on body weight for the Mid- and Low-group animals (see Report 9.3.3., Table 3, and Appendix 4.2.1).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects in the mean daily food consumption in any test item-treated group when compared to the control.

Statistically significant changes (decreases at p<0.05 or p<0.01) in the daily food consumption were seen in the Low-, Mid- and High-dose parental males at some evaluated periods during study. However, there was no clear tendency or dose response, and the mean values of the test item-treated groups calculated for the total treatment period differed less than 4% from the control, thus the obtained values had no biological relevance. Therefore, these facts were not considered as a test item-related effect.

Similar picture was seen in case of the females. Statistically significant changes (decreases or increases at p<0.05 or p<0.01) in the daily food consumption were seen in the Low-, Mid- and High-dose parental females at some evaluated points during the pre-mating period, but there was no tendency or dose response. Those facts were not considered to be a test item-related effect. In the gestation period, decreased food consumption values with some occasional statistical significance were recorded in the test item-treated groups. In the lactation period, no substantial differences between groups were seen, although the mean value calculated for the period showed slightly higher increase (significant at p<0.01) for the High-dose group. But the general trend of this period where a near zero growth is expected showed no test item effect.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
When compared to the controls, there were no differences in any haematological parameters that could be considered toxicologically significant in any test item-treated group of Cohort-1A males and females, although statistically significant in some cases: decreased mean cell haemoglobin and MCC (p<0.05 or p<0.01) in the Mid- and High-dose males, increased platelet count (p<0.05) in High-dose females, and decreased relative amount of reticulocytes in the Mid- and High-dose females (p<0.05 and p<0.01, respectively) (see Report, Table 24, and Appendix 4.4.3). However, all those values did not suggest a dose response, while the same time were within the normal physiological and historical control range.

There was no effect of the test item on blood-clotting parameters. No statistically significant or biologically relevant changes in Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PTT) were recorded for any dose groups of parental (F0) animals.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes on the serum chemistry parameters of the animals in Cohort 1A, although certain parameters showed statistically significant differences when compared to the controls: decreased cholesterol concentration in the Mid- and High-dose males (p<0.01), decreased albumin concentration and albumin/globulin ratio in High-dose females (p<0.05 or <0.01, respectively).

Cholesterol: A similar decrease but without statistical significance was seen in the High-dose females (see Report, Table 25, and Appendix 4.4.3). However, the observed mean values were within the normal physiological range (mean of historical control ± 2-times standard deviation, SD) in all cases (males and females).

Protein: Decreased values for parameters related to protein content (total protein concentration, albumin concentration and albumin/globulin ratio) were observed in High-dose females, the difference from control was statistically significant for albumin and A/G ratio (p<0.05 or <0.01, respectively. However, no similar trends were seen in High-dose males. Furthermore, all the observed values were within the normal physiological range. Therefore, the observed differences between values were considered as having no biological relevance (the actual values of female control animals were higher than usual based on the historical control database) and not being related to test item administration.

No statistically significant changes were recorded for any other parameters in the test item-treated parental males and females when compared to control.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in the test item-treated groups were observed in the urinalysis.

No biologically relevant changes were recorded in the volume, pH or specific gravity of the urine samples between the control and any test item-treated groups. Although the mean total volume in the Low-dose males was higher than in the control, and the mean total volume in High-dose females was lower than in the control. But due to the lack of statistical significance and lack of similar trends in the two sexes, and as all the individual values were within the historical control range, these findings were not considered as a toxicologically relevant, test item-related effect. The appearance of urine samples (including the colour and amount of urine crystals or bacteria) as well as the urinary glucose, ketones, nitrite, blood, protein, bilirubin and urobilinogen observed in the test item treated groups was comparable to controls.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant increases considered test item-related were observed in the liver weights in both sexes in the High-dose. These differences were supported by test item-related histopathological findings.
There was an increase in absolute and relative (to body and brain) liver weights in the females, and body-adjusted liver weights in the males. In the females, absolute liver weights were statistically significantly higher by 19.2% (p<0.01), body-related by 23.1% (p<0.01), and by 18.8% (p<0.01) when brain-related. In the males, liver weights adjusted to body weight were increased by 23.5%. The absolute and brain-related weights were not statistically different (at 11.4% and 11.5% above control, respectively).

Male thymus weights showed an apparent statistically significant difference (decrease) in absolute weight in the High-dose, but this was considered to be a consequence of the body weights reduced by treatment. There was a decrease in absolute weight (up to -20%) and relative to body weight (-10.8%) and to brain (-19.5%). While thymus weights were also decreased in the females, statistical significance was not reached, absolut or adjusted to body weight. Occasionally, small thymus was recorded in the High-dose males at necropsy, with microscopic decreased size/cellularity of thymic cortex in both sexes. However, this was attributed to the body weight difference.

Other statistical differences in the organ weights of the High-dose group of parental (F0) generation were attributed to differences in body weight or were small differences not considered to be related to treatment: brain, kidneys, thyroid and testes weight (relative to body) in High-dose males, and heart weight (absolute and relative to brain) in High-dose females.

There were no statistical differences in organ weights in the Mid- or Low-dose groups that were considered to be treatment-related.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Animal found dead: One male in the High-dose group was found dead on Day 36. A cause of death was not determined for this animal. No test item-related changes were seen. At macroscopic evaluation, the cannibalisation of numerous organs/tissues was observed (heart, lungs, trachea, lacrimal glands, mandibular lymph nodes, salivary glands, spinal cord, thymus, skin (fore limbs), thyroid/parathyroid glands and larynx/pharynx).

Terminally euthanised animals: At macroscopic evaluation, treatment-related changes were recorded in the liver and thymus of the High-dose group (considered to be non-adverse). Enlargement of the liver appeared in 1/23 High-dose males, and 10/24 High-dose females. Pale lobes (multifocal) were detected in 4/24 High-dose females. Enlarged liver was also seen in 2/24 Mid-dose females. Enlargement of the liver was present in 4/24 Low-dose female (In three animals, not verified by histopathology). Small thymus was recorded in 1/24 High-dose female.

All other changes, like small spleen in one Mid-dose female and red discolouration of a High-dose male, were considered to be incidental or a common background.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Animal found dead: During microscopic evaluation, minimal diffuse mixed infiltration of the sinusoids and slight centrilobular (macrovesicular) vacuolation in the liver were noted. These changes were regarded as incidental or background.

Terminally euthanised animals: During histopathology evaluation, treatment-related microscopic findings were noted in the liver and thymus in examined animals of the High-dose group.

In the liver, mixed form of hepatocellular vacuolation was described in High-dose males and females. This form of vacuolation was different from macrovesicular feature occurring in controls as predominantly centrilobular and periportal zonation were seen. The severity ranged from minimal to moderate in the males, and minimal to severe in the females. The incidence was 14/23 males and 21/24 females of the High-dose group.

In the thymus, a slight decrease in cortical size / cellularity was present in 1/23 High-dose male and 1/24 High-dose female. These changes were reported to be a consequence of body-weight decreases due to treatment mentioned above; however, the overall low incidence in both sexes cannot completely exclude occurrence by chance in these two cases.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Reproductive organs: All other changes were considered to be incidental, or a common background.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No effects related to test-item treatment were noted during gestation, parturition or the post-partum period.

The mean length of gestation was 22.80, 22.43, 22.62 and 22.39 days in Control, Low-, Mid- and High-dose groups, respectively. These values are well within the normal range for this strain of rat and indicated no effect of test item on this parameter (despite occasional statistical significance).

As far as it could be observed during the study, the parturition was normal for all animals.
The number of corpora lutea and number of implantation sites was comparable to the control mean in all dose groups, no statistically significant differences were noted.

There were no significant differences or effects that could be ascribed to treatment on the pre-implantation, intrauterine, post-natal or total mortality values (litter mean and %) at up to the High-dose level.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Sperm motility and morphology as well as number of sperms were examined in the study for parental males. There was no treatment-related effect at any dose levels of the parental (F0) generation.

In case of sperm motility, the observed values in the Low-dose group were comparable to those of the control level. The number of immotile sperms slightly increased in the Mid-dose males and High-dose males, but without statistical significance. There was no dose response, and the observed values were in harmony with the historical control range, thus these facts were considered as not related to the test-item treatment.

There were no biologically relevant changes in the sperm morphology. The ratio of abnormal sperms was significantly increased in the Low- and Mid-dose groups (p<0.01 and p<0.05, respectively); but the level in the High-dose group was comparable to the control, and all data was in the normal range. Based on the lack of dose response, these values were not considered to be a test item-related effect.

No effect was seen on the number of sperms (either in testis or in cauda epididymis) in the animals of the High-dose groups when compared to the Control.
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no significant differences between the Control and test item-treated groups with regard to reproductive ability, mating fertility or gestation.

The mating indices were normal in all test item-treated groups (100%) for both males (see details in Table X) and females. The fertility index was 92-100% in all male and female test item-treated groups, while the control value was slightly lower (83%). The gestation index for females was 100% in the Control group, and test item-treated groups also showed values (95-96%) within the normal range. Hence, no test item-related differences were noted for these parameters.

Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm-positive vaginal smears and/or vaginal plugs) occurred within up to 5 days of pairing (cohabitation) in each case. Extreme values were only detected in the Low-dose group in three cases (6 days) and in High-dose group on one occasion (7 days), but the latter female previously had prolonged diestrus).

The mean duration of mating was 2.50, 3.42, 2.33 and 2.75 days in the Control, Low-, Mid- and High-dose groups, respectively. Due to the lack of dose response within the normal range, the observed statistical significance (p<0.05) in the Low-dose group was not considered to be a test item-related effect.
See under "General Toxicity"
Key result
Dose descriptor:
NOAEL
Remarks:
(1500 mg/kg diet, nominal, highest level tested)
Effect level:
124 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity
Key result
Dose descriptor:
NOAEL
Remarks:
(500 mg/kg diet, nominal)
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: Systemic toxicity
Key result
Critical effects observed:
no
Reproductive function: sperm measures:
not examined
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Based on the external evaluation, pups were generally normal, there were no adverse effect of treatment.
One pup in the Control group and two pups in the Low-dose group had missing tail tip. Two of them were cannibalised / found dead by PND1.
Haemorrhage was recorded on PND 0 for one Low-dose pup on the abdominal area and one Mid-dose pup on both cheeks, but all those animals became symptom-free by the next day.
Several pups of a litter in the Mid-dose group had thin fur in the period of PND12-15.
None of these events were considered to be related to the test item treatment.

Cohort 2A (dedicated to developmental neurotoxicity testing at young adult age) - No test item-related clinical signs were detected for any animals of this cohort. Minor clinical signs were recorded for some animals as follows: Piloerection was recorded for a High-dose male in the period of PND 80-87, while red discharge from left eye was noted for another High-dose male in the period of PND 29-75. All females of this cohort were symptom-free during the entire observation period.

Cohort 2B (dedicated to developmental neurotoxicity testing at age of weanlings (all animals were terminated on PND 22)) - All the animals (males and females) included in this cohort were symptom-free during the entire observation period.

Cohort 3 (dedicated to developmental immunotoxicity testing) - All the animals (males and females) included in this cohort were symptom-free during the entire observation period.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on the mortality and viability of pups on PND 0 and PND 4.
The number of stillborn / cannibalised pups or dead (born alive) pups and number of living pups at the end of lactation period (PND 21) as well as the number of affected litters were comparable to the control in all cases, indicating no test item-related effect.
The number of living pups, dead pups, survival index and sex ratio were monitored during the lactation period (PND 0, PND 4, PND 7, PND 14 and PND 21). The number of viable pups as well as viability (survival) indices in the test item-treated groups were comparable to the relevant control values in all test item-treated groups of F1 generation.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean individual birth weights of the live-born pups were within the normal range, with 6.48 to 6.58 g across all treated groups vs. 6.72 g in the control group, males and females combined). During lactation ( PND 0 to PND 21), there was evidence of significant test material-related growth retardation in the High-dose group, both sexes (p<0.01), the mean body weights at PND21 as well as the body-weight gains from PND0 through PND21 remained about 10% behind the controls for both, males and females (see Report 9.4.2, Table 17, and Appendix 4.7.1).
However, since the High-dose P0 dams were also smaller than the controls, and the offspring was fed via the dams´ milk, the difference in pup weights cannot be a direct effect on weight development but rather may be attributed to a sub-optimal physical constitution of the dams.

Cohort 1A (this cohort was used for the OECD 408 study)
There was no test item-related effect on the Low-dose group animals. There was no effect on body-weight growth of males at any dose level. Females of the High-dose group had a lower starting body weight at the high dose (and to some extent also at the mid dose), and they had a lower growth rate compared to controls at the Mid- and High-dose level.

Although a statistically significant decrease (by 7%, p<0.05) was observed in the body weight at the end of the evaluation period (PND 120) for High-dose males, this was not considered as clear evidence of test item effect, as the starting body weight at weaning (on PND 21) also showed a similar difference (approx. 8%, p<0.05). As no significant difference was seen in the body weight gain of the High-dose males during the PND 21-120 period, the observed values were considered as showing no test item related effect. Neither were there significant changes on body weight in the Mid- and low-dose groups.

However, in case of Cohort 1A females, the original difference in the starting body weight of Mid- and High-dose animals compared to control (by 4.9% and 9.0%) became wider by the end of the evaluation period (9.0% in case of Mid-dose females and 18.1% in case of High-dose females, both significant at p<0.01). Furthermore, the body weight gain during this period (PND 21-120) was statistically significant in the Mid- and High-dose females (10.3% and 20.3%, p<0.01 in both cases) compared to the control group. These values indicated a test item-related effect in the females of the Mid- and High-dose groups, although the impact of a lower starting body weight is difficult to evaluate. There were no significant changes in the Low-dose females of this cohort.

Cohort 2A - Lower body weight (by 9% and 15% on PND 70) was seen in Mid- and High-dose males, respectively. The difference was statistically significant at p<0.05 and p<0.01. Lower body weight compared to control (by 17%, significant at p<0.01 level) was also recorded in High-dose females of this cohort.

Cohort 2B - There was no statistically significant difference in body weight between control and test item-treated groups, although a slight decrease was observed in the High-dose group compared to control (by 10% in males and by 8% in females on PND 22).

Cohort 3 - The body weight profile showed no significant difference between control and test item-treated groups, although slight decrease was observed in the High-dose group compared to control (by 6% in males and by 8% in females on PND 56).
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Cohort 1A (this cohort was used for the OECD 408 study) - No test item-related changes were noted at ophthalmoscopy examination. All examined animals were found to be normal even at early time-point (PND 36-42) as well as before termination (PND 101-110).
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A (this cohort was used for the OECD 408 study) - No toxicologically relevant changes were seen in any treated group for any of the haematological parameters evaluated. Statistically significant differences were seen in some cases (decreased mean cell haemoglobin and MCC (p<0.05 or p<0.01) in Mid- and High-dose males, increased platelet count (p<0.05) in High-dose females, and decreased relative amount of reticulocytes in Mid- (p<0.05) and High- (p<0.01) dose females). However, all those values were within the normal physiological and historical control range. There was no dose response. Therefore, these observations were considered to be caused by biological variability, and therefore not treatment-related. There was no effect of the treatment on blood-clotting parameters. No statistically significant or biologically relevant changes in Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PTT) were recorded for any dose groups of parental (F0) animals.

Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A (this cohort was used for the OECD 408 study) - There were no treatment-related changes on the serum chemistry parameters of the parental (F0) generation. Decreased cholesterol concentration was observed in the High-dose males, the difference being statistically significant (p<0.01) when compared to control animals. A similar, but slight (without statistical significance) decrease was also observed in High-dose females. However, the observed mean values were within the normal physiological range (mean of historical control ± 2SD) in all cases. The differences were relatively small, and from this data alone, would have been considered to be unrelated to treatment. In this study the P0 generation also had a similar slightly lower cholesterol values, hence a relationship with treatment cannot be totally excluded, but the degree of change was small and all values were in the normal range. The differences observed in Cohort 1A were considered to be of equivocal relationship with treatment, but in the absence of any other associated findings, such a small difference was not considered to be an adverse treatment-related finding.

Decreased values for parameters related to protein content (total protein concentration, albumin concentration and albumin/globulin ratio) were also observed in High-dose females, the difference from control being statistically significant (p<0.05 or p<0.01) for albumin and A/G ratio. However, no similar trends were seen in High-dose males (actually in High-dose males an opposite change, statistically significant increase (p<0.05) in total protein content was detected). Furthermore, all the observed values were within the normal physiological range (mean of historical control ± 2SD). Therefore, the observed differences between values were considered as having no biological relevance (the actual values of female control animals were higher than usual based on the historical control database) and not treatment-related.

Statistically significant changes (at p<0.05 level) were recorded for the concentrations of some ions: increased sodium and calcium concentration were noted for High-dose males, and increased chloride concentration was noted in High-dose females when compared to control animals. However, the differences were minor (less than 5%), no similar trends were seen in the other sex in any of those cases, furthermore all those data were within the normal physiological control and/or historical control range. Therefore, they were considered as animal variability, not related to the test material. No relevant changes were recorded for any other parameters in the test material-treated parental males and females when compared to control.
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
The sex ratio did not differ significantly from control in any dose groups at any occasions.

Cohort 1A (this cohort was used for the OECD 408 study): Additional parameters (balanopreputial separation for males, vaginal opening and oestrus cycle for females) were also examined to follow the sexual development of F1 weanlings. Slightly slower development in the High-dose males was observed; however, this was considered to be secondary to low body weight and not effect of exposure. Vaginal patency (vaginal opening) evaluated for all female pups selected for this cohort daily beginning on PND 22 and no effect on this parameter was observed in the Low-, Mid- and High-dose groups of Cohort 1A females. Additionally, the oestrous cycles did not show significant differences between the groups.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A (this cohort was used for the OECD 408 study) - Terminal body weights of males and females were statistically significantly lower in the High-dose group, and considered to be treatment-related; decreases were -7.2% (p<0.05) and -18.8% (p<0.01) for the males and females, respectively. A treatment-related decrease of terminal body weights (by 9.0%, p<0.01) was also observed in Mid-dose females. No changes in terminal body weights were observed in Mid-dose males or Low-dose animals when compared to the controls.

Statistically significant differences, considered test item-related, were present in the liver weights in both sexes of the High dose group. These differences were supported by test item-related histopathological findings. There was an increase in absolute and relative (to body and brain) liver weights in the High-dose males, and for body weight-adjusted liver weights in the High-dose females. In the High-dose males, absolute liver weights were statistically significantly higher by 20.1% (p<0.01), body-related by 29.6% (p < 0.01), and by 19.9% (p < 0.01) when brain-related. In High-dose females, liver weights adjusted to body weight were increased by 15.0% (p < 0.01) but values were slightly lower than control for absolute and brain-adjusted weights. A statistically significant increase considered test item-related was defined in the liver of Mid-dose males. When related to body and to brain weights, liver weights were larger by 11.5% (p<0.01) and by 10.8% (p <0.05), respectively. No statistically significant differences were reported in Mid-dose female livers.

In both sexes of the High-dose group, the treatment-related lower body weight had a consequence with some statistically significant lower organ weights. But based on microscopic examination these organ weight differences were considered as secondary changes due to the body weight effect, without evidence of a direct effect of the test item. These statistical differences were in High-dose males: brain, kidney and thyroid (only when adjusted to body weight); and in High-dose females: brain, adrenals, thyroid, uterus (adjusted to body weight), heart, kidney, spleen and ovary. Other statistical differences in females were not attributed to an adverse effect, were lower and higher kidney weights for absolute or brain-adjusted and body-adjusted; spleen, ovaries and mandibular lymph nodes absolute and adjusted to brain. In these cases, histological examination established that the organs were normal. In addition, the treatment-related lower body weight in females had a consequence on brain weight adjusted to body weight, and on lower absolute or brain-related heart and mandibular lymph node weights. But based on microscopic examination these differences were considered as secondary changes due to the body weight effect, without evidence of a direct effect of the test item.

There were no significant differences considered to be treatment-related in the Low-dose group (males and females) compared to controls.

Cohort 2A - The only organ weight measured in this cohort was the brain. Slightly higher brain weights in both sexes when adjusted for body weight, and lower in females for absolute weights were detected in the High- dose when compared to control. These statistical differences were considered to be secondary to body weight and not an effect of the test item. There were no statistically significant differences in the Mid-or Low-dose groups.

Cohort 2B - The brain weight was similar in the control and test item-treated groups in male and female rats.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A (this cohort was used for the OECD 408 study) - Treatment-related changes were recorded in the liver of High-dose males. No treatment-related changes were noted in the High-dose females. Pale mottled/diffuse discoloration was seen in 4/22 males (#8001, #8002, #8017 and #8020) of the High-dose only (a similar incidence of this finding as in the parental (F0) generation animals). All other changes were incidental, a common background or related to the oestrous cycle.

Cohort 1B (this cohort was used for the OECD 408 study) - At necropsy, a total of 2/18 High dose males had hepatic changes, pale and enlarged liver was seen in 1/18 and pale liver in 1/18 males and liver weights were slightly increased in the High dose only. All other changes were considered to be incidental, or a common background. In recovery subset animals there were no significant liver weight or any hepatic effects observed, and no other no treatment-related changes were detected at necropsy.

Cohort 2A - No treatment-related changes were detected in any animals of any dose groups of this cohort. Depressed area of the left cerebrum was noted in one Control female (#5558), but it was an incidental finding.

Cohort 2B - No abnormalities in the brain were recorded for any animals in any dose groups during macroscopic evaluation.

Cohort 3 - Organs and tissues related to the immune system were closely examined for this cohort. Macroscopic evaluation did not show any abnormalities: spleen, thymus, femur with bone marrow, sternum with bone marrow, axillary and mesenteric lymph nodes, Peyer’s patches were normal in all test item-treated groups.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related histopathological findings were also noted in examined High-dose animals. There was a mixed form of hepatocellular vacuolation observed in the livers of High-dose males and females: centrilobular (mainly) and periportal zonation were seen. The severity varied from minimal to moderate with frequency of 19/22 males and 13/24 females. A severe intensity with diffuse distribution altered liver was found in 1/22 males. No similar vacuolation was recorded in controls. This observation was considered to be non-adverse.

In addition to the standard histopathology examination, for F1 parental females only, a quantitative assessment of primordial follicles and corpora lutea was conducted. There were no meaningful differences in total number (left and right ovaries) of primordial follicles and corpora lutea between Control and High Dose females. All other changes were incidental, a common background or related to the oestrous cycle.

Cohort 1B (this cohort was used for the OECD 408 study) - No treatment-related microscopic changes were observed in examined males and females. There was hepatocellular vacuolation of the liver recognised in 2/5 males dosed at a concentration level of 1500 ppm; however, macrovesicular forms were demonstrated and considered to be a common background observations similar to those commonly seen in control animals. Hence, it was considered there was no effect in the recovery animals. In addition, a quantitative assessment of primordial follicles and corpora lutea was conducted in the females according to the Study plan. There were no meaningful differences in total number (left and right ovaries) of primordial follicles and corpora lutea between Control and High-dose females.

Cohort 2A:
Neurohistopathology of CNS - During neurohistopathology examination of the central nervous system (CNS), no treatment-related changes were seen in the neuroanatomical subsites of the central nervous system (Levels 1-9) in the examined animals of the High-dose group.

Neurohistopathology of PNS - During neurohistopathology evaluation of the peripheral nervous system (PNS), no treatment-related changes were seen in the examined animals of the High-dose group. Minimal focal/multifocal unilateral axonal degeneration of the sciatic nerve was noted in 2/12 High-dose males and 1/12 High-dose females. Minimal focal/multifocal unilateral axonal degeneration of the sural nerve was seen in 1/12 Control male and 2/12 Control females. Minimal focal/multifocal axonal degeneration of the spinal cord (lumbar region) was seen in 1/12 Control male and 2/12 High-dose males and 2/12 Control female and 1/12 High-dose females. Slight neuronal vacuolation of the subiculum was defined in 1/12 High-dose males. However, all these findings were regarded as incidental or a common background, not related to test item administration.

Brain morphometrics - At morphometric evaluation of the brains of Cohort 2A rats, lower hippocampus thickness values were observed in High-dose males, and lower cornu Ammonis thickness values were observed in High-dose females (both when measured unilateraly). Biological significance of these changes should be interpreted along with the histological examination and clinical data.

Cohort 2B:
Neurohistopathology of CNS: During neurohistopathology examination no treatment-related changes were seen in the neuroanatomical subsites of the central nervous system (Levels 1-9) in the examined animals of any dose groups. An incidental finding of slight focal acute intramyelinic oedema of cingulate cortex in a High-dose female was observed, but based on the isolated incidence, it was not considered to be a treatment-related effect.

Brain morphometrics - At morphometric evaluation of the brains, there were no relevant differences in test item-treated groups when compared to control rats of this cohort when the following parameters were evaluated: cortex thickness, caudate-putamen width, corpus callosum, dentate gyrus, Cornu Ammonis, hippocampus thicknesses and cerebellum height.
Other effects:
no effects observed
Description (incidence and severity):
Anogenital Distance: Anogenital distance was measured on PND 0 in new-born F1 pups. There was no treatment-related effect on this parameter in the treated males and females of the F1 generation.

Sperm analysis: Sperm motility and morphology as well as number of sperms were examined in the study for Part 2 males (Cohort 1A (This cohort was used for the OECD 408 study). No treatment-related effect was seen in any dose groups of this cohort.

Thyroid hormone analysis: No toxicologically relevant changes in the test item-exposed groups were observed in the thyroid hormone values of Cohort 1A.

A statistically higher value (p<0.05) was recorded for the High-dose females for T4 (of 45.5 pg/mL). However, the Parental T4 range (47.9 - 60.8 pg/mL for animals of approximately the same age) shows that the control mean for the 1A group was relatively low. The High-dose female 1A data is not higher than the normal range, hence the statistical difference is not considered to be of biological significance.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Cohort 1A (this cohort was used for the OECD 408 study) - There were no test item-related effects in the neurological assessment, as there were no observed differences in animal behaviour, general physical condition or in the reactions to different types of stimuli in the control or test item-treated groups.

Cohort 2A - There were no test item-related effects in the neurological assessment, as there were no observed differences in animal behaviour, general physical condition or in the reactions to different types of stimuli in the control or test item-treated groups.

Cohort 2A: Auditory startle reflex - Auditory startle reflex was examined at PND 24/25. There were no treatment-related effects detected on the evaluated parameters (maximum amplitude of the response, area under the response curve, onset latency and duration of the response).
Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
Cohort 1A (this cohort was used for the OECD 408 study): Immunophenotyping - The immunophenotyping analysis conducted to determine the relative and absolute counts of six different lymphocyte subpopulation indicated that there was no shift in the immunological steady state distribution of "helper" (CD4+) or cytotoxic (CD8+) thymus derived lymphocytes or natural killer (NK) cells, related to the treatment. In case of absolute counts, no statistical difference was observed between treated groups and the control group for any parameters in either sex. In case of relative counts, the Dunnett’s post-hoc test revealed that no statistical difference was observed between the treated groups and the control group for any parameters in either sex of this cohort.

Cohort 3: TDAR Assay - No statistically or biologically significant changes were detected in the anti-SRBC IgM level of the test item-treated groups of male animals. Decreased anti-SRBC IgM levels compared to the negative control group were detected in the Low- and High-dose groups of the female animals (the difference was statistically significant at p<0.05 level in case of High-dose females). However, the observed values were in line with the level observed in the male negative control animals (the statistical analysis showed no significant difference when High-dose females were compared to the male Control group), and there was no dose response. Based on historic control data, males and females are expected to have similar titres, so it is considered scientifically valid to use the 2 sexes in evaluation potential treatment relationships. The difference was mainly caused by two Control females having extreme anti-SRBC antibody level, the same situation was observed in the Mid-dose group showed also extremely high anti-SRBC IgM). Thus no test item effect was concluded in the TDAR assay.

Treatment with the positive control substance (cyclophosphamide) gave the anticipated strong inhibitory response: the decrease in the anti-SRBC IgM level of the positive control animals compared to the negative control group was statistically significant in male and female animals, thus indicating impairement ot the functional immune response.
Key result
Dose descriptor:
NOAEL
Remarks:
(highest dose tested) (1500 mg/kg diet, nominal)
Generation:
F1
Effect level:
ca. 130 mg/kg bw/day
Based on:
test mat.
Remarks:
actual dose received through P0 dams
Sex:
male/female
Basis for effect level:
other: sexual differentiation during lactation phase
Key result
Dose descriptor:
NOAEL
Remarks:
(500 mg/kg diet, nominal)
Generation:
F1
Effect level:
42 mg/kg bw/day
Based on:
test mat.
Remarks:
actual dose received through P0 dams
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other:
Remarks:
This cohort was used for the OECD-408 study.
Key result
Dose descriptor:
LOAEL
Remarks:
(1500 mg/kg diet)
Generation:
F1
Effect level:
130 mg/kg bw/day
Based on:
test mat.
Remarks:
actual dose received through P0 dams
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other:
Remarks:
This cohort was used for the OECD-408 study.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Table 9. Summary of analytical results (concentration and homogeneity of the test item in the diet)

Batch

Group

Percentage of target concentration measured for each batch (%)

Before
feeding

Middle
of batch use

Near end of batch use

1st

150 ppm

97.1%

99.2%

-

89.1%

500ppm

92.8%

97.6%

-

95.0%

1500ppm

96.8%

92.3%

-

88.5%

2nd

150 ppm

104.9%

101.2%

93.1%

95.7%

500ppm

102.4%

100.9%

90.9%

94.2%

1500ppm

94.8%

104.1%

86.0%

87.2%

3rd

150 ppm

93.0%

105.4%

-

95.2%

500ppm

98.3%

95.9%

-

96.4%

1500ppm

87.2%

90.9%

-

94.0%

4th

150 ppm

99.2%

102.2%

-

-

500ppm

103.2%

106.2%

-

-

1500ppm

92.7%

96.7%

-

-

5th

150 ppm

101.6%

-

-

-

500ppm

102.2%

(not fed to rats)

-

-

1500ppm

95.5%

(not fed to rats)

-

-

Table 10. Mean test item intake (see Report, Table 3)

Nominal concentration in diet

(ppm)

150

500

1500

Target dose levels

(mg/Kg bw/day)

10

33

100

Samples

Mean Test Item Intake (mg/Kg bw/day)

Parental
(F0) generation

Males

9.14

30.19

93.87

Females

14.25

47.43

149.81

Combined

11.92

39.57

124.29

Cohort 1A

(F1 generation)

- used for the OECD 408 study

Males

11.09

37.52

115.64

Females

12.56

42.05

128.78

Combined

11.82

39.79

122.21

Cohort 1B

(F1 generation)

- used for the OECD 408 study

Males

10.07

33.90

103.57

Females

11.72

40.63

125.24

Combined

10.90

37.26

114.40

Cohort 2A

(F1 generation)

Males

13.64

43.43

138.17

Females

14.59

47.92

157.85

Combined

14.12

45.68

148.01

Cohort 2B

(F1 generation)

Males

24.57

73.02

194.74

Females

24.38

75.53

199.54

Combined

24.47

74.28

197.14

Cohort 3

(F1 generation)

Males

15.51

52.35

159.70

Females

17.12

53.81

166.67

Combined

16.32

53.08

163.19

Overall Mean Intake
For F1 Generation

12.58

41.91

130.25

Overall Mean Intake
(of both generations)

12.42

41.32

128.77

Note: Total combined value is the mean daily intake of test item from diet, with the parental and cohort means (for both sexes) weighted for the duration of food measurements for those groups. For this purpose, the mean values of 99 and 118 days were used for parental males and females; 100, 116, 69, 1 and 41 days were used for Cohorts 1A, 1B, 2A, 2B and 3, respectively.

Table 11. Selected body weight parameters of parental animals

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated males

24

24

24

23

 

Male, Body weight on Day 71 (g)

570.6

532.2*

537.5*

519.4**

DN

difference (%)

-6.7

-5.8

-9.0

Male, Body weight on Day 95 (g)

588.8

547.2*

555.8

535.9**

DN

difference (%)

-7.1

-5.6

-9.0

 

Male, Body weight gain Day 1-95 (g)

321.9

282.6*

291.9

273.0**

DN

difference (%)

-12.2

-9.3

-15.2

 

Number of evaluated females

24

24

24

24

 

Female, Body weight on Day 71 (g)

304.5

293.5

289.5*

275.9**

DN

difference (%)

-3.6

-4.9

-9.4

 

Female, Body weight gain Day 1.71 (g)

113.0

105.2

97.2**

85.1**

DN

difference (%

-6.9

-14.0

-24.7

 

Female, Body weight on GD20 (g)

455.0

444.5

432.2*

413.7**

DN

difference (%)

-2.3

-5.0

-9.1

 

Female, Body weight gain GD0-20 (g)

147.7

140.9

140.1

130.5**

DU

difference (%)

-4.6

-5.1

-11.6

 

Female, Body weight on PPD21 (g)

353.3

346.2

350.2

334.6**

DN

difference (%)

-2.0

-0.9

-5.3

 

Female, Body weight gain PPD0-21 (g)

-3.8

-6.5

12.3*

15.7**

DN

difference (%)

72.8

-427.6

-517.4

 

Notes: Data (group mean values) were rounded to one decimal place.

Statistical significance compared to control: *: atp<0.05, **: at p<0.01

DN: Dunnett’s Multiple Range Test; DU: Dunn test; NS: Statistically not significant when compared to control

Table 12. Selected food consumption parameters of parental animals

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated males

24

24

24

23

 

Male, Food consumption (Day 1-95) (g/day)

28.490

28.01

27.37

27.67

NS

difference (%)

-1.7

-3.9

-2.9

Number of evaluated females

24

24

24

24

 

Female, Food consumption (Day 1-71) (g/day)

18.77

18.85

18.77

19.26

NS

difference (%)

0.4

0.0

2.6

 

Female, Food consumption (GD0-20) (g/day)

25.23

24.37

23.15**

22.54**

DN

difference (%)

-3.4

-8.2

-10.7

Female, Food consumption (PPD0-21) (g/day)

65.4

62.84

63.99

59.92**

DU

difference (%)

-3.9

-2.2

-8.4

 

Notes: Data (group mean values of daily food consumption) were rounded to two decimal places.

GD: Gestation Day

Statistical significance compared to control: *: atp<0.05, **: at p<0.01

DN: Dunnett’s Multiple Range Test; DU: Dunn test; NS: Statistically not significant when compared to control

Table 13. Summary of selected haematology parameters (parental animals)

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

Male, Red Blood Cell Counts (M/μL)

8.381

8.664

9.048**

8.752

DU

difference (%)

3.4

8.0

4.4

 

Male, Relative amount of LUC (%)

1.500

0.740**

0.840*

1.230

DN

difference (%)

-50.7

-44.0

-18.0

 

Female, Red Blood Cell Counts (M/μL)

8.027

8.260

8.063

7.974

NS

difference (%)

2.9

0.4

-0.7

 

Female, Relative amount of LUC (%)

0.430

0.280

0.460

0.370

NS

difference (%)

-34.9

7.0

-14.0

 

Notes: Data (group mean values, n=10) were rounded to two or three decimal places.

LUC: Large Unstained Cells

Statistical significance compared to control: *: atp<0.05, **: at p<0.01

DU: Dunn Test, DN: Dunnett’s Multiple Range Test, NS: Statistically not significant compared to control

Table 14. Selected clinical chemistry parameters of parental animals

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Male, Cholesterol (mmol/L)

1.968

1.998

1.789

1.620*

DN

difference (%)

1.5

-9.1

-17.7

Male, Total protein (g/L)

55.060

55.510

56.160

56.490

NS

difference (%)

0.8

2.0

2.6

 

Male, Albumin (g/L)

30.010

29.590

30.480

30.190

NS

difference (%)

-1.4

1.6

0.6

 

Male, A/G ratio

1.200

1.130

1.190

1.140

NS

difference (%)

-5.8

-0.8

-5.0

 

Female, Cholesterol (mmol/L)

1.901

1.890

1.861

1.660

NS

difference (%)

-0.6

-2.1

-12.7

 

Female, Total protein (g/L)

59.890

59.390

59.100

56.280*

DN

difference (%)

-0.8

-1.3

-6.0

Female, Albumin (g/L)

34.050

34.180

33.160

30.360**

DN

difference (%)

0.4

-2.6

-10.8

 

Female, A/G ratio

1.330

1.340

1.290

1.170*

DN

difference (%)

0.8

-3.0

-12.0

 

Notes: Data (group mean values) were rounded to three decimal places.

A/G ratio: Albumin/globulin ratio

Statistical significance compared to control: *: at p<0.05, **: at p<0.01

DN: Dunnett’s Multiple Range Test, NS: Statistically not significant when compared to control

Table 15. Summary of reproductive parameters of parental males

Parameters

Dose groups

Control

Low dose

Mid dose

High dose

Number of treated animals

24

24

24

24

Number of pre-terminal death

0

0

0

1

Number of males used for mating

24

24

24

23

Number of successful mating

24

24

24

23

Number of infertile animals

4

0

2

0

Male mating index (%)

100

100

100

100

Male fertility index (%)

83

100

92

100

Note: Due to pre-terminal death of #4019 male in the High dose group, the animal #4018 was used for mating of two females (#4518 and #4519).

Table 16. Summary of reproductive parameters of parental females

Parameters

Dose groups

Control

Low dose

Mid dose

High dose

Number of treated animals

24

24

24

24

Number of pre-terminal death

0

0

0

0

Number of females used for mating

24

24

24

24

Number of sperm positive females

24

24

24

24

Number of females with no implantation sites

4

0

2

0

Number of pregnant females

20

24

22

24

Number of pregnant females with live born(s)

20

23

21

23

Number of pregnant females not delivered

0

1

1

1

Female mating index (%)

100

100

100

100

Female fertility index (%)

83

100

92

100

Female gestation index (%)

100

96

95

96

Notes: Four females in the Control group and two females in the Mid-dose group were not pregnant. One female in the Low-, Mid- and High-dose groups had complete intrauterine mortality.

Table 17. Summary of the intrauterine evaluation

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated females

24

24

24

24

 

Mean number of corpora lutea

13.58

14.79

13.79

15.38

NS

Mean number of implantations

13.08

14.29

13.21

14.25

NS

Pre-natal mortality, mean

0.70

1.13

1.41

1.13

NS

Pre-natal mortality (%), mean

5.45

12.10

13.71

12.52

NS

Number of pups born, mean

15.05

13.58

13.18

13.25

NS

Number of live born pups, mean

15.00

13.21

13.00

13.13

NS

Post-natal mortality PND0-21, mean

0.00

0.04

0.00

0.00

NS

Post-natal mortality PND 0-21 (%), mean

0.00

0.29

0.00

0.00

NS

Total mortality until PND21, mean

1.10

1.88

1.68

1.33

NS

Total mortality until PND21 (%), mean

8.00

17.02

15.59

13.96

NS

Number of living pups on PND21, mean

9.60

8.88

9.27

9.17

NS

Duration of pregnancy (days)

22.80

22.43*

22.62

22.39*

D

Notes: Mean values were rounded to two decimal places. Four females in the Control group and two females in the Mid-dose group were not pregnant. Furthermore, three females across the treated groups did not deliver. They were not included in the mortality evaluation.

Statistical significance compared to control: *: at p<0.05, **: at p<0.01

D: Duncan’s Multiple Range Test, NS: Statistically not significant compared to control

Table 18. Sperm analysisof parental males

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of sperm analysed, mean

200

200

200

200

NS

Number of not motile sperms, mean

35.98

35.42

43.65

42.96

NS

Number of abnormal shaped sperm, mean

1.04

1.75**

1.54*

0.93

D

Weight of testis (g)

2.031

n.d.

n.d.

1.981

NS

Total number of sperms in testis (x106)

183.85

n.d.

n.d.

183.70

NS

Weight ofcauda epididymis (g)

0.31

n.d.

n.d.

0.31

NS

Total number of sperms incauda epididymis(x106)

86.04

n.d.

n.d.

86.11

NS

Notes: For sperm analysis, samples of all males were processed for sperm morphology and motility analysis (in duplicates). Samples of all animals in the Control and High-dose groups were used for enumeration.

Statistical significance compared to control: *: p<0.05; **: p<0.01

D: Duncan’s Multiple Range Test, NS: statistically not significant, n.d.: no data (not analysed)

Table 19. Organ weight data of parental males

 

Organ weight

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated animals

24

24

24

23

 

Terminal body weight, g

586.9

545.5*

552.4

531.7**

DN

(difference %)

-7.0

-5.9

-9.4

 

Liver (absolute), g

16.818

15.924

17.224

18.740

NS

(difference %)

-5.3

2.4

11.4

 

Liver (relative to body), %

2.858

2.907

3.108

3.528**

DN

(difference %)

1.7

8.8

23.5

 

Liver (relative to brain), %

741.71

705.82

761.47

827.29

NS

(difference %)

-4.8

2.7

11.5

 

Thymus(absolute), g

0.464

0.425

0.383

0.373*

DN

(difference %)

-8.4

-17.4

-19.7

 

Thymus (relative to body), %

0.078

0.077

0.069

0.070

NS

(difference %)

-1.0

-11.3

-10.8

 

Thymus (relative to brain), %

20.45

18.79

17.02

16.45*

DN

(difference %)

-8.1

-16.7

-19.5

 

Notes: Organ weight data (group mean values) were rounded to two or three decimal places.

Statistical significance compared to control: *: atp<0.05, **: at p<0.01

DN: Dunnett’s Multiple Range Test, NS: Statistically not significant compared to control

Table 20. Organ weight data of parental females

 

Organ weight

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated animals

20

23

21

23

 

Terminal body weight, g

328.8

325.6

324.0

318.5

NS

(difference %)

-1.0

-1.4

-3.1

 

Liver (absolute), g

15.355

16.263

15.675

18.303**

DN

(difference %)

5.9

2.1

19.2

 

Liver (relative to body), %

4.667

4.986

4.839

5.743**

DN

(difference %)

6.8

6.8

23.1

 

Liver (relative to brain), %

757.78

798.44

752.50

900.46**

DN

(difference %)

5.4

-0.7

18.8

 

Thymus(absolute), g

0.262

0.263

0.253

0.227

NS

(difference %)

0.6

-3.5

-13.2

 

Thymus (relative to body), %

0.079

0.081

0.078

0.071

NS

(difference %)

1.8

-1.6

-10.6

 

Thymus (relative to brain), %

12.92

12.88

12.15

11.15

NS

(difference %)

-0.3

-6.0

-13.7

 

Notes: Organ weight data (group mean values) were rounded to two or three decimal places.

Statistical significance compared to control: *: atp<0.05, **: at p<0.01

DN: Dunnett’s Multiple Range Test, NS: Statistically not significant compared to control

Table 21. Summary of mortality data of F1 generation

Parameters

Dose groups

Control

Low dose

Mid dose

High dose

Total number of pups born

301 / 20

326 / 23

290 /21

318 /23

Number of stillborn or cannibalized pups

1 / 1

11 / 7

4 / 3

4 / 4

Number of live born pups

300 / 20

315 / 23

286 / 21

314 / 23

Died at birth (born alive)

0 / 0

0 / 0

0 / 0

0 / 0

Cannibalized during PND 0-21 (born alive)

8 / 7

14 / 7

5 / 4

2 / 2

Found dead during PND 0-21 (born alive)

0 / 0

2 / 2

1 / 1

2 / 2

Total number of dead pups during PND 0-21

8 / 7

16 / 9

6 / 4

4 / 3

Total number of pups culled on PND4

100 / 20

86 / 23

78 /21

90 /23

Number of living pups on PND 21

192 / 20

213 / 23

202 / 21

220 / 23

Notes: Mortality numbers mean number of pups / number of affected litters.

Table 22. Body weight data (offspring)

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated litters

20

23

21

23

 

Mean litter body weight (PND0), g

6.715

6.481

6.575

6.536

NS

Mean body weight of male pups (PND0), g

6.894

6.726

6.742

6.647

 

Mean body weight of female pups (PND0), g

6.535

6.258

6.420

6.387

 

Mean litter body weight (PND21), g

67.038

65.311

64.268

60.414

 

Mean body weight of male pups (PND21), g

68.618

67.086

65.957

61.878

 

Mean body weight of female pups (PND21), g

65.238

63.543

62.535

58.814

 

Mean body weight gain per litter (PND0-21), g

60.311

58.783

57.703

53.882

 

Mean body weight gain of male pups (PND0-21), g

61.740

60.298

59.248

55.217

NS

Mean body weight gain of female pups (PND0-21), g

58.665

57.253

56.118

52.434

NS

Female, Body weight on PPD21 (g)

353.3

346.2

350.2

334.6**

DN

Notes: Data (group mean values) were rounded to three decimal places. By the end of lactation period, there was no surviving male pups in litters #2522 and #4508.

DN: Dunnett’s Multiple Range Test; NS: Statistically not significant compared to control

Table 23. Selected body weight parameters of Cohort 1A animals (used for the OECD 408 study)

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated males

20

23

21

22

 

Male, Body weight on PND21 (g)

68.7

67.5

64.9

63.1**

D

difference (%)

-1.8

-5.5

-8.2

 

Male, Body weight on PND120 (g)

595.3

610.3

584.6

555.3*

DN

difference (%)

2.5

-1.8

-6.7

 

Male, Body weight gain PND21-120 (g)

526.6

542.8

519.7

492.2

NS

difference (%)

3.1

-1.3

-6.5

 

Number of evaluated females

22

23

21

24

 

Female, Body weight on PND21 (g)

65.8

62.9

62.6

59.9**

D

difference (%)

-4.5

-4.9

-9.0

 

Female, Body weight on PND120 (g)

331.3

312.9

301.5**

271.4**

DN

difference (%)

-5.6

-9.0

-18.1

 

Female, Body weight gain PND21-120 (g)

265.5

250.0

238.9**

211.5**

DN

difference (%)

-5.8

-10.0

-20.3

 

Notes: Data (group mean values) were rounded to one decimal place.

Statistical significance compared to control: *: atp<0.05, **: at p<0.01

D: Dunn test; DN: Dunnett’s Multiple Range Test, NS: Statistically not significant when compared to control

Table 24. Summary of selected haematology parameters (Cohort 1A parental animals - used for the OECD 408 study)

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Male, Mean Cell Haemoglobin (pg)

18.06

17.35*

17.30*

17.22**

DU

difference (%)

-3.9

-4.2

-4.6

 

Male, MCHC (g/dL)

33.88

33.05

32.76**

32.63**

DU

difference (%)

-2.4

-3.3

-3.7

 

Male, Platelet count (K/μL)

965.8

995.6

990.5

984.8

NS

difference (%)

3.1

2.6

2.0

 

Male, Relative amount of reticulocytes (%)

2.420

2.409

2.471

2.382

NS

difference (%)

-0.5

2.1

-1.6

 

Female, Mean Cell Haemoglobin (pg)

18.63

18.49

18.40

18.10

NS

difference (%)

-0.8

-1.3

-2.9

 

Female, MCHC (g/dL)

33.27

33.58

33.15

33.08

NS

difference (%)

0.9

-0.4

-0.6

 

Female, Platelet count (K/μL)

862.6

895.5

965.2

959.0*

DN

difference (%)

3.8

11.9

11.2

 

Female, Relative amount of reticulocytes (%)

2.60

2.42

2.30*

2.033**

DU

difference (%)

-7.0

-11.4

-21.8

 

Notes: Data (group mean values, n=20-24) were rounded as appropriate (to 1-3 decimal places).

Statistical significance compared to control: *: atp<0.05, **: at p<0.01

DU: Dunn Test, DN: Dunnett’s Multiple Range Test, NS: Statistically not significant compared to control

 

Table 25. Selected clinical chemistry parameters of Cohort 1A animals (used for the OECD 408 study)

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Male, Cholesterol (mmol/L)

2.190

1.930

1.758**

1.669**

DN

difference (%)

-11.8

-19.7

-23.8

 

Male, Total protein (g/L)

57.92

58.19

58.90

60.18*

DN

difference (%)

0.5

1.7

3.9

 

Male, Albumin (g/L)

31.59

31.77

32.12

33.01

NS

difference (%)

0.6

1.7

4.5

 

Male, A/G ratio

1.20

1.20

1.20

1.22

NS

difference (%)

-0.4

0.4

1.9

 

Male, ALKP (U/L)

102.7

92.8

90.5

105.6

NS

difference (%)

-9.6

-11.8

2.9

 

Male, ALT / GPT (U/L)

43.5

47.9

52.8

52.9*

DU

difference (%)

10.0

21.3

21.6

 

Male, Bile acid (μmol/L)

12.770

12.867

14.974

16.295*

DU

difference (%)

0.8

17.3

27.6

 

Female, Cholesterol (mmol/L)

2.408

2.117

2.111

1.943

NS

difference (%)

-12.1

-12.3

-19.3

 

Female, Total protein (g/L)

67.05

64.49

65.93

64.51

NS

difference (%)

-3.8

-1.7

-3.8

 

Female, Albumin (g/L)

41.50

39.35

40.05

37.99*

DN

difference (%)

-5.2

-3.5

-8.5

 

Female, A/G ratio

1.636

1.565

1.548

1.442**

DN

difference (%)

-4.3

-5.4

-11.9

 

Female, ALKP (U/L)

52.9

50.6

62.6

73.4**

DU

difference (%)

-4.3

18.4

38.7

 

Female, ALT / GPT (U/L)

46.9

52.6

55.5

50.8

NS

difference (%)

12.2

18.5

8.3

 

Female, Bile acid (μmol/L)

14.050

16.408

16.636

17.875*

DU

difference (%)

16.8

18.4

27.2

 

Notes: Data (group mean values, n=20-22) were rounded as appropriate (to 1-3 decimal places).

ALT (GPR): Alanine Aminotransferase (Glutamate-pyruvate transaminase); ALKP: Alkaline phosphatase

Statistical significance compared to control: *: at p<0.05, **: at p<0.01

DN: Dunnett’s Multiple Range Test; DU: Dunn test; NS: Statistically not significant when compared to control

Table 26. Organ weight data of Cohort 1A males (used for the OECD 408 study)

 

Organ weight

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated animals

20

23

21

22

 

Terminal body weight, g

571.2

584.9

560.1

530.1*

DN

(difference %)

2.4

-1.9

-7.2

 

Liver (absolute), g

15.824

16.906

17.300

19.003**

DN

(difference %)

6.8

9.3

20.1

 

Liver (relative to body), %

2.770

2.889

3.089**

3.589**

DU

(difference %)

4.3

11.5

29.6

 

Liver (relative to brain), %

692.08

739.60

767.05*

829.84**

DN

(difference %)

6.9

10.8

19.9

 

Notes: Organ weight data (group mean values) were rounded to two or three decimal places.

Statistical significance compared to control: *: atp<0.05, **: at p<0.01

DN: Dunnett’s Multiple Range Test, DU: Dunn’s test, NS: Statistically not significant compared to control

 

Table 27. Organ weight data of Cohort 1A females (used for the OECD 408 study)

 

Organ weight

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated animals

22

23

21

22

 

Terminal body weight, g

317.1

298.4

288.6**

257.7**

DN

(difference %)

-5.9

-9.0

-18.8

 

Liver (absolute), g

10.223

9.906

9.680

9.570

NS

(difference %)

-3.1

-5.3

-6.4

 

Liver (relative to body), %

3.234

3.328

3.365

3.717**

DU

(difference %)

2.9

4.1

15.0

 

Liver (relative to brain), %

497.73

481.71

473.12

471.77

NS

(difference %)

-3.2

-4.9

-5.2

 

Notes: Organ weight data (group mean values) were rounded to two or three decimal places.

Statistical significance compared to control: *: atp<0.05, **: at p<0.01

DN: Dunnett’s Multiple Range Test, DU: Dunn’s test, NS: Statistically not significant compared to control

Table 28. Immunophenotyping of Cohort 1A (absolute counts)

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Males

 

Number of analysed samples

20

21

21

22

NS

Number of splenocytes (cell/μL)

206.9

178.6

241.0

240.9

NS

Number of B cells (cell/μL)

84.2

75.5

103.8

98.6

NS

Number of NK cells (cell/μL)

8.6

7.6

7.5

10.5

NS

Number of NKT cells (cell/μL)

11.0

9.1

13.8

11.0

NS

Number of T cells (cell/μL)

79.8

64.6

85.0

86.4

NS

Number of Helper T cells (cell/μL)

54.4

42.5

61.0

56.2

NS

Number of Cytotoxic T cells (cell/μL)

18.8

14.8

20.0

21.3

NS

Females

 

Number of analysed samples

22

21

21

24

NS

Number of splenocytes (cell/μL)

203.9

176.7

228.7

237.8

NS

Number of B cells (cell/μL)

83.6

77.0

96.2

102.5

NS

Number of NK cells (cell/μL)

8.4

6.5

8.0

8.5

NS

Number of NKT cells (cell/μL)

10.5

8.5

13.3

9.6

NS

Number of T cells (cell/μL)

75.0

66.2

83.9

87.1

NS

Number of Helper T cells (cell/μL)

50.6

45.5

61.1

59.1

NS

Number of Cytotoxic T cells (cell/μL)

17.3

14.6

18.5

20.1

NS

Notes: Data (group mean values) are rounded to one decimal place.

NS: statistically not significant, according to the decision tree indicated in Appendix 10 (relevant phase report), where Dunnett’s test is used for significance.

 

Table 29. Immunophenotyping of Cohort 1A (relative counts)

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Males

 

Number of analysed samples

20

21

22

22

NS

Number of B cells (cell/μL)

40.5

41.1

42.6

40.2

NS

Number of NK cells (cell/μL)

4.2

4.4

3.3

5.3

NS

Number of NKT cells (cell/μL)

5.4

5.2

6.6

4.9

NS

Number of T cells (cell/μL)

39.0

36.9

35.5

36.5

NS

Number of Helper T cells (cell/μL)

68.2

66.2

91.8

65.4

NS

Number of Cytotoxic T cells (cell/μL)

22.9

22.4

32.9

23.9

NS

Females

 

Number of analysed samples

22

21

21

24

NS

Number of B cells (cell/μL)

40.2

42.7

41.6

42.8

NS

Number of NK cells (cell/μL)

4.3

3.7

3.7

3.8

NS

Number of NKT cells (cell/μL)

5.2

4.9

6.5

4.2

KW*

Number of T cells (cell/μL)

37.5

38.2

36.6

36.9

NS

Number of Helper T cells (cell/μL)

66.8

68.3

89.7

67.7

NS

Number of Cytotoxic T cells (cell/μL)

23.2

22.3

27.5

23.2

NS

Notes: Data (group mean values) are rounded to one decimal place.

NS: statistically not significant, according to the decision tree indicated in Appendix 10 (relevant phase report), Dunnett’s test for significance.

Table 30. Selected body weight parameters of Cohort 2A

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated males

10

12

11

12

 

Males, Body weight on PND70 (g)

467.3

448.8

426.2*

397.8**

DN

Males, Body weight gain PND21-70 (g)

 

 

 

 

 

Number of evaluated females

12

12

11

12

 

Females, Body weight on PND70 (g)

271.9

261.4

267.6

224.5**

DN

Females, Body weight gain PND21-70 (g)

 

 

 

 

 

Notes: Data (group mean values) are rounded to one decimal place.

Statistical significance compared to control: *: at p<0.05, **: at p<0.01

DN: Dunnett’s Multiple Range Test; NS: Statistically not significant when compared to the control.

Table 31. Selected organ weight parameters of Cohort 2A

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated males

10

12

11

12

 

Males, Brain weight (g)

2.094

2.064

2.129

2.089

NS

Males, Brain/Body weight

0.416

0.419

0.452

0.482**

DU

Number of evaluated females

12

12

11

12

 

Females, Brain weight (g)

1.901

1.871

1.917

1.803*

DN

Females, Brain/Body weight

0.665

0.677

0.701

0.759**

DN

Notes: Data (group mean values) are rounded to three decimal places.

Statistical significance compared to control: *: at p<0.05, **: at p<0.01

DN: Dunnett’s Multiple Range Test; DU:Dunn’s test,NS: Statistically not significant when compared to the control.

Table 32. Selected morphometric parameters of Cohort 2A

 

Parameters

Dose groups

 

Males

Females

 

Control

High dose

Control

High dose

 

Number of evaluated animals

 

 

 

 

 

Level 3, cortex thickness (1 & 5)

3649.9

3673.5

3519.8

3463.0

NS

Level 3, cortex thickness (2 & 6)

3749.7

3740.5

3314.6

3206.1

NS

Level 3, caudate-putamen width

6725.8

6634.7

6125.9

5901.0

NS

Level 3,corpus callosum thickness

702.4

722.4

650.6

691.8

NS

Level 4, dentate gyrus thickness

628.7

602.4

589.6

567.7

NS

Level 4, Cornu Ammonis thickness

651.7

627.1

646.1

579.0**

DN

Level 4, hippocampus thickness

1497.7

1428.7*

1365.2

1315.64

DN

Level 7,cerebellum height

4897.3

4715.3

4489.4

4308.8

NS

Notes: Data (group mean values) are rounded to one decimal place.

Statistical significance compared to control: *: atp<0.05, **: at p<0.01

DN: Dunnett’s Multiple Range Test; NS: Statistically not significant when compared to the control.

Table 33. Selected morphometric parameters of Cohort 2B

 

Parameters

Dose groups

 

Males

Females

 

Control

High dose

Control

High dose

 

Number of evaluated animals

11

13

11

12

 

Level 3, cortex thickness (1 & 5)

4271.1

4295.4

4048.0

4033.0

NS

Level 3, cortex thickness (2 & 6)

4171.5

4110.0

3829.4

3941.5

NS

Level 3, caudate-putamen width

6803.5

6508.1

6411.9

6374.8

NS

Level 3,corpus callosum thickness

567.5

546.2

530.0

598.3

NS

Level 4, dentate gyrus thickness

597.7

567.9

570.0

584.0

NS

Level 4, Cornu Ammonis thickness

659.3

656.9

618.3

625.1

NS

Level 4, hippocampus thickness

1428.6

1471.6

1390.7

1381.7

NS

Level 7,cerebellum height

5051.8

5134.9

4956.3

4902.0

NS

Notes: Data (group mean values) are rounded to one decimal place.

NS: Statistically not significant when compared to the control.

Table 34. Mean anti-SRBC IgM concentration of Cohort 3

Group Number / Designation

Number of
animals (n)

Measured IgM concentration
(Mean± standard deviation)

(u/mL)

log (ln)
transformed

Cohort 3 males

1.   Negative control

11

5899.3 ± 5926.0

8.32 ± 0.84

2.   Low dose

12

6068.4 ± 4138.1

8.50 ± 0.68

3.   Mid dose

11

5040.7 ± 3319.5

8.33 ± 0.69

4.   High dose

12

3583.1 ± 1452.8

8.12 ± 0.38

5.   Positive control

10

108.5** ± 34.5

4.64** ± 0.33

Cohort 3 females

1.  Negative control

11

12097.4 ± 14509.5

8.93 ± 0.96

2.   Low dose

12

5478.6 ± 3617.7

8.47 ± 0.50

3.   Mid dose

11

14998.3 ± 12980.6

9.34 ± 0.81

4.   High dose

12

4106.1* ± 3710.7

8.06 ± 0.73

5.   Positive control

10

89.4** ± 22.1

4.47** ± 0.21

Notes: Data (group mean values) are rounded to one decimal place.

Statistical significance compared to control: *: atp<0.05, **: at p<0.01(Dunn test or Dunnett’s Multiple Range Test)

Table 35. Summary of thyroid hormone analysis (Cohort 1A)

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Male, T4 (ng/mL)

56.25

51.91

51.97

56.20

NS

difference (%)

-7.7

-7.6

-0.1

 

Male, TSH (pg/mL)

1847.8

1689.7

2169.1

2414.1

NS

difference (%)

-8.6

17.4

30.6

 

Male, Terminal body weight (g)

571.2

584.9

560.1

530.1*

DN

difference (%)

2.4

-1.9

-7.2

 

Male, Thyroid & parathyroid weight (g)

0.0322

0.0345

0.0325

0.0347

NS

difference (%)

7.5

1.2

7.9

 

Female, T4 (ng/mL)

33.97

38.89

33.45

45.45*

DN

difference (%)

14.5

-1.5

33.8

 

Female, TSH (pg/mL)

2118.7

1505.0

1441.9

2606.9

NS

difference (%)

-29.0

-31.9

23.0

 

Female, Terminal body weight (g)

317.1

298.4

288.6**

257.7**

DN

difference (%)

-5.9

-9.0

-18.8

 

Female, Thyroid & parathyroid weight (g)

0.0250

0.0259

0.0235

0.0242

NS

difference (%)

3.6

-6.1

-3.1

 

Notes: Data (group mean values, n=10) were rounded to one to four decimal places

Conclusions:
Based on the results observed in this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity (parental generation) was determined to be 500 mg/kg diet (40 mg/kg bw/day) and the NOAEL for reproductive toxicity (parental generation) was determined to be 1500 mg/kg diet (124 mg/kg bw/day).
 
The NOAEL for systemic toxicity and physical development (100-day - F1 generation) was determined to be 500 mg/kg diet (42 mg/kg bw/day), while the NOAELs for sexual development of the F1 pups was determined to be 1500 mg/kg diet (130 mg/kg bw/day).
Additionally, in this study, the test material was not observed to exhibit any neurotoxic, immunotoxic, or endocrine disrupting effects.
Executive summary:

A key combined extended one-generation reproductive toxicity (EOGRT) / sub-chronic oral toxicity study was conducted according to Guideline recommendations (OECD 443 / OECD 408) to evaluate specific life stages not covered by other types of toxicity studies and to test for effects that may occur as a result of pre- and post-natal chemical exposure to the test material (Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol (OAPP), EC# 700 -960 -7). The OECD 443 study was combined with the OECD 408 (90-day rat) study using the F1 generation (hence exposure was in utero to weaning, then a further approximately 90 days), to evaluate sub-chronic toxicity of the test material.

 

The study was divided in two experiments: Part 1 and Part 2.

In Part 1 of the study, the test material was administered continuously in the diet to male rats (24/concentration) for a period of 10 weeks (pre-mating), through the mating period and after that until they were no longer needed for assessment of reproductive effects for a total of at least 13 weeks of treatment at concentrations of 0, 150, 500, or 1500 mg/kg diet. Female rats (24/concentration) were treated for 10 weeks pre-mating and during the mating, gestation, and lactation periods until they were no longer needed for assessment of reproductive effects for a total of at least 16 weeks of treatment at concentrations of 0, 150, 500, or 1500 mg/kg diet (equivalent to an overall mean intake (for both sexes combined) of 0, 12, 40, and 124 mg/kg bw/day, respectively). The day of birth (i.e. when parturition was complete) was defined as post-partum day 0 (PPD 0). Male and female pups born in Part 1 were used for Part 2 Cohort 1A (This cohort was used for the OECD 408 sub-chronic toxicity evaluation study.). After weaning (on PND 21), animals (at least 20/sex/concentration) were treated for 100 days. Recovery animals, Control and High-dose groups, located within Cohort 1B was also used for the OECD 408 study: In the end, 5/sex/concentration were kept for 30 additional days for recovery after the treatment had finished. During this period they were fed the study control diet. Animals of cohorts 2A (neurobehavioral testing - at least 10/sex/concentration), 2B (neurohistopathology assessment - at least 10/sex/concentration), and Cohort 3 (developmental immunotoxicity - at least 10/sex/concentration ) were treated the same way as Part 2 (Cohort 1A) animals.

 

Parental (F0) generation

No adverse clinical signs of toxicity were observed. Mortality was observed in one High-dose male but considered as background finding, and not related to any systemic effects of the test material.

Treatment-related changes on body weight / body weight gain were seen in the High-dose group (1500 mg/kg diet) which were considered to be adverse. Small differences in the Mid-dose (500 mg/kg diet) and Low-dose group (150 mg/kg diet) were not considered to represent an adverse effect.

No treatment-related changes were noted in reproductive parameters during mating and gestation, delivery and post-partum/lactation periods in any of the treatment groups. There were no effects of treatment observed on F1 offspring viability, clinical signs, or development and gross necropsy did not reveal any remarkable findings. Haematology and clinical chemistry parameters evaluated were unaffected by treatment.

Reductions in some organ weights were observed, considered as a consequence of lower body weights and not an adverse effect (thymus, spleen, heart, kidney, prostate and/or ovary) in the High-dose group (1500 mg/kg diet). Histopathology showed the organs to be normal; or in the case of thymus, a very low incidence of decreases in cortical size / cellularity was compatible with being secondary to lower body weight. Higher liver weights associated with necropsy observations of enlarged and/or pale appearance, and hepatocellular vacuolation (mixed form) with centrilobular and periportal zonation was observed in the High-dose group, ascribed to the test material in the diet. The severity ranged from minimal to moderate in High-dose males, and minimal to severe in High-dose females. Vacuolation corresponded with statistically significant increase in absolute and relative (to body and brain) liver weights in the females, and body-adjusted liver weights in the male. The vacuolation and other hepatic observations were considered to be non-adverse effects.

 

Cohorts 1A and 1B (these cohorts were used for the OECD 408 study)

There were no mortalities after weaning, no adverse clinical signs and no effects on neurotoxicity endpoints.

There was no statistically significant, test item-related effect on the Low-dose F1A-animals. There was a slightly significant decrease (about 7%, p<0.05) in the body weight of the High-dose F1A-males (at PND120), while the terminal body-weight gains (PND21-120) remained insignificant for all male groups. On the other hand, the terminal mean body weights of females of the Mid- and High-dose group were statistically lower than those of the controls (-9 and -18 %, respectively), more marked for the mean body-weight gains (-10% and -20%, respectively (from PND21-120) (see Report Table 20). The decreases in mean body weight in the male and female High-dose groups are considered to be treatment-related.

There were no adverse effects on haematology or clinical chemistry in any group. Reductions in some organ weights were observed, considered as a consequence of lower body weights and not an adverse effect (as was seen in the F0 animals).

Treatment-related mixed form of hepatocellular vacuolation occurred in the High-dose males and females, visualised as pale lobes seen in some males at necropsy. A statistically significant liver weight increase, considered treatment-related, was present in both sexes in the High-dose, and to a slight extent in Mid-dose males. Centrilobular (mainly) and periportal zonation were seen at histopathology; similar to the parental animals, the severity varied mainly from minimal to moderate and there were proportional frequencies viewed in the male and females. The changes (qualitatively and quantitatively similar to F0 generation) were considered to be non-adverse hepatic changes.

 

In the recovery animals of the 90-day toxicity study part (F1 generation), there were no significant differences in liver weights and no treatment-related macroscopic or microscopic hepatic changes in examined males and females after the recovery period; this was considered to confirm the non-adverse nature of the terminal hepatic observations.

 

Additionally, a quantitative assessment of primordial follicles and corpora lutea conducted in terminal and recovery females did not show any meaningful differences in total number of primordial follicles and corpora lutea between Control and High dose females.

 

Cohort 2A

There were no mortalities after weaning, no adverse clinical signs and no effects on neurotoxicity endpoints. Body weight differences were similar to those in the cohort 1A and 1B. There were no adverse effects on brain weight. In Cohort 2A of the F1 generation, no gross treatment-related changes were detected in the liver at any dose levels. No treatment-related findings were seen in detailed histopathological examination of the CNS and PNS of examined animals in the High-dose group. Brain morphometrics showed no clear adverse effects.

 

Cohort 2B

There were no mortalities after weaning, no adverse clinical signs, no significant body weight differences and no organ weight differences. In Cohort 2B of F1 generation, no treatment-related macroscopic changes in any organs or tissues, and no histopathological changes were seen in the CNS of the examined animals in the High-dose group. Brain morphometrics showed no differences between groups.

 

Cohort 3

There were no mortalities after weaning and no adverse clinical signs. There were no statistically significant differences in body weight. In Cohort 3 of F1 generation, no macroscopic findings were observed in the macroscopically observed organs and tissues related to the immune system at any dose level. No treatment-related effect was observed in the functional immunological assessment (TDAR) assay.

Based on the results observed in this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity (parental generation) was determined to be 500 ppm (equivalent to 40 mg/kg bw/day) and the NOAEL for reproductive toxicity (parental generation) was determined to be 1500 ppm (124 mg/kg bw/day).

The oral NOAEL for systemic toxicity physical development (100-day - F1 generation) was determined to be 42 mg/kg bw/day (equivalent to 500 mg/kg diet), while the NOAELs for sexual development of the F1 pups were determined to be 500 ppm 130 mg/kg bw/day (equivalent to 1500 mg/kg diet) respectively.

Additionally, in this study, the test material was not observed to exhibit any neurotoxic, immunotoxic, or endocrine disrupting effects.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
Combined with the Extended One-Generation Reproduction Toxicity Study (EOGRTS), OECD 443: F1-genenration used for Repeated dose study according to OECD 408.
Principles of method if other than guideline:
The study was combined with the Extended One-Generation Reproduction Toxicity Study (EOGRTS), OECD 443: Male and female F1 pups were used for this OECD 408 study: After weaning (on PND 21), animals were treated for 100 days. Recovery animals (Control and High-dose groups) of this F1-generation were derived from Cohort 1B and kept for 30 additional days after the treatment had finished.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Test material form:
liquid: viscous
Details on test material:
- Manufacturers identification: Novares LA 300
- Substance type: organic
- Test material is 'Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol' (OAPP), EC list number 700-960-7 (assigned manually to validated substances from inquiries by ECHA). Originally the substance phenol, methylstyrenated, CAS No. 68512-30-1, EC No. 270-966-8 was submitted for registration. Subsequent to substance validation, the identity of the substance was changed by ECHA.
- for additional information see respective study record
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: RÜTGERS Novares GmbH; Batch no. 38900
- Composition of test material: composition is specified in IUCLID Sect. 13 - Assessment reports under Certificate of Analysis_Novares LA 300_phenol, methylstyrenated
- Expiration date of the lot/batch: 13 February 2018
- Purity test date: 2015-03-24

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, <70 RH%), protected from light
- Stability under test conditions: Not specified

FORM AS APPLIED IN THE TEST (if different from that of starting material): Light yellow to colourless liquid, at low temperature tendency to crystallisation

OTHER SPECIFICS:
- Purity: 100% (UVCB)
- Name of test material (as cited in study report): Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol, OAPP

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI
Details on species / strain selection:
The rat is regarded as suitable species for toxicology and reproduction studies. The Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. The same strain was used in the previous experimental work of the project performed at the Test Facility.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
(Further details see IUCLID entry 7.81)
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Address: Sandhofer Weg 7, D-97633 Sulzfeld, Germany) from SPF colony
Animals of F1 generation were group-housed (2 animals of the same sex per cage) after the cohort selection, wherever possible. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: no

DETAILS OF FOOD AND WATER QUALITY: tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15.2 – 27.0°C (target range: 22 ± 3°C)
- Humidity (%): 20 – 74% (target range: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light (from 6.00 a.m. to 6.00 p.m.)

IN-LIFE DATES: From: 2016-10-27 To: 2017-06-14

Administration / exposure

Route of administration:
oral: feed
Vehicle:
corn oil
Remarks:
used as the vehicle to facilitate mixing of the test item into the diet to obtain the desired dietary concentration.
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Each diet concentration was prepared as one batch of 100-120 kg/concentration level at the first preparation time, and variable amounts at the further preparation time (30-200 kg/concentration level), depending on the available stability data and/or the expiry date of the diet.
- Mixing appropriate amounts with (Type of food): The test material (OAPP) was incorporated into ssniff® SM R/M-Z+H “Complete Feed for Rats and Mice-maintenance” by ssniff Spezialdiäten GmbH (D-59494 Soest, Germany) to generate the test concentrations required for the study (150, 500 and 1500 ppm). Test material (OAPP) was incorporated into the diet using a solution in corn oil and mixed for up to approximately 12 minutes (approximately 6 minutes for premix preparation, and 6 minutes for preparation of the complete diets). The diets were always produced in an ascending order regarding the test item concentration. Similar diet preparation procedures were used to generate control diet (0 mg OAPP/kg diet).
- Storage temperature of food: Prepared diets were stored at room temperature under dry conditions in sewed bags pending and during transport to CiToxLAB Hungary Ltd. At CiToxLAB Hungary Ltd., the prepared diet bags were stored in areas designated for diet storage at room temperature (approximately 15-21°C), under dry conditions, pending transfer to animal room at approximately 22 ± 3°C for animal feeding.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn Oil (Corn oil was used as the vehicle to facilitate mixing of the test item into the diet to obtain the desired dietary concentration) supplied by Henry Lamotte GmbH (Bremen, Germany)
- Concentration in vehicle: 4%
- Lot/batch no. (if required): Batch Numbers: 16-3329 (8001046002) / 16-3424 (8001213001), 16-3481 (8001409001) / 17-3551 (8002635001); Expiry dates: November 2017 / March 2018 / May /2018 / July 2018)
- Purity: A quality of Ph.Eur.8 (2014) & BP 2016
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the diets for homogeneity and concentration of OAPP was performed at the designated Test Site (Test Site #1) using a validated GC method [6]. Test-item containing diet samples of an appropriate weight were collected from at least five different places of the diet container (in duplicates) from each dose group at least before the start, in the middle and near the end of the use of each batch of diets during the study to determine concentration and homogeneity. At least one sample (in duplicate) was taken from the control diet for concentration analysis at each occasion.
Duration of treatment / exposure:
100 days
Frequency of treatment:
continuous in the diet
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Control
Dose / conc.:
150 ppm
Remarks:
Low Concentration (150 mg/kg diet), corresponds to ca. 12 mg/kg bw/day (F1 from PND21-PND121, measured)
Dose / conc.:
500 ppm
Remarks:
Intermediate Concentration (500 mg/kg diet nominal) corresponds to ca. 40 mg/kg bw/day (measured, F1 from PND21-PND121)
Dose / conc.:
1 500 ppm
Remarks:
High Concentration (1500 mg/kg diet nominal), corresponds to ca. 122 mg/kg bw/day (measured, F1 from PND21 to PND121)
No. of animals per sex per dose:
- At least 20 animals/sex/concentration of the F1 generation (Cohort 1A) from the OECD 443 study with which the 408 was combined
- 30-day recovery groups (Cohort 1B) - 5 animals/sex in the Control and High-dose group
Control animals:
yes, concurrent vehicle
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels (equivalent to dietary concentrations of 150, 500 and 1500 mg/kg diet) were selected by the Sponsor based on available data and information from previous experimental work, including the available results of a preliminary Dose Range Finding (DRF) palatability study (Study code: 15/094-220PE) and an OECD No. 422 study ((Study code: 15/094-220P) performed at the Test Facility.

In the OECD No. 422 study, OAPP was administered in diet at the concentrations of 300, 1250 and 5000 ppm, (equivalent to dietary doses of approximately 24.5, 97.1 and 337.6 mg/kg bw/day in the Low-, Mid- and High-dose groups, respectively) to Wistar rats for at least 28 days. Administration of OAPP caused significantly reduced body weights, reduced body-weight gain and reduced food consumption in the High-dose of both sexes; slight differences were seen in the Mid-dose females.

Organ weight and macroscopic observations at necropsy showed hepatic enlargement in both sexes at the High-dose and in Mid-dose males. At histopathology evaluation, hepatocellular vacuolation was also detected confirming the macroscopic observations. However, in the absence of effects on the liver function (as indicated by the clinical chemistry parameters) the hepatic changes were considered as probably an adaptive rather than an adverse effect. Based on this it was decided to add 30 day recovery groups to the OECD 408 study, to confirm this is an adaptive response. The No-Adverse-Effect Level (NOAEL) of OAPP for systemic, reproductive and developmental toxicity was concluded to be1250 ppm (97.1 mg/kg bw/day).

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Adult animals inspected for signs of morbidity and mortality twice daily, at the beginning and end of the working day as practical.

DETAILED CLINICAL OBSERVATIONS: Yes (see Report, Appendix 4.1.3)
- Time schedule: Detailed clinical observations were made on all adult animals at least prior to the first treatment (to allow for within-subject comparisons) and weekly thereafter. These observations were made outside the home cage in a standard arena, at similar times, generally during the morning. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards). Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes (see Report, Table 20, and Appendix 4.2.3)
- Time schedule for examinations: Body weight was recorded with a precision of 1 g at weaning (PND 21, prior to the start of treatment which was designated as Day 1 of individual treatment), then at least weekly, including on Day 100 (last treatment day) and prior to necropsy (if scheduled necropsy, fasted, on Day 101).

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 (Food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g twice per week. Weekly food consumption was calculated for reporting purposes. Determination of food consumption of Cohort 1A of F1 generation was performed twice per week. The first measurement of given food was made at weaning (PND 21, i.e. Day 1). The remaining, non-consumed food was weighed twice per week with a precision of 1 g. Food hoppers were replenished more frequently if required.

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: Yes
- Time schedule for examinations: Ophthalmoscopic examination was conducted in all animals of Cohort 1A soon after the start of treatment (Week 6) and in the Control (Group 5) and High-dose (Group 8) animals, during Week 12/13 of the treatment. As no treatment-related alterations were found, no additional animals of Low- (Group 6) and Mid- (Group 7) dose groups or recovery animals were examined near/at termination.
- Dose groups that were examined:all animals of Cohort 1A. All animals of Control (Group 5) and High-dose (Group 8) Groups.

HAEMATOLOGY: Yes (Report, Table 24, and Appendix 4.4.3)
- Time schedule for collection of blood: Prior to scheduled necropsy
- Anaesthetic used for blood collection: Yes (pentobarbital anaesthesia) - After an overnight period of food deprivation of animals (fasting), blood samples were collected by heart puncture under pentobarbital anaesthesia, for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), for blood clotting times (in tubes with sodium citrate as anticoagulant)
- Animals fasted: Yes (overnight)
- How many animals: all surviving animals

CLINICAL CHEMISTRY: Yes (Report, Table 25, and Appendix 4.4.3)
- Time schedule for collection of blood: Prior to scheduled necropsy
- Animals fasted: Yes (overnight)
- How many animals: all surviving animals

URINALYSIS: Yes (Report, Appendix 4.4.3)
- Time schedule for collection of urine: Prior to scheduled necropsy (collected overnight (for approximately 16 hours))
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (with no access to food, but with access to water)

NEUROBEHAVIOURAL EXAMINATION: Yes (Report, Table 22, and Appendix 4.1.5)
- Time schedule for examinations: Towards the end of the treatment period, during Week 12/13, but not earlier than in Week 11
- Dose groups that were examined: each animal of Cohort 1A which was used to populate the OECD 408 study
- Battery of functions tested: sensory activity / grip strength / motor activity / other: functional observation battery, including measurements of the landing foot splay and fore/hind limb grip strength; and motor activity assessment.

IMMUNOLOGY: For the investigation of possible pre- and post-natally induced immunotoxic effects, Cohort 1A animals were subjected to extra steps at termination including weighing of extra organs/tissues associated with the immune system and splenic lymphocyte subpopulation analysis.

THYOID HORMONE ANALYSIS: Blood sampling was done for thyroid hormone analysis (TSH, T4 and/or T3) of Cohort 1A animals (see Report, Table 26
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy (scheduled on PND111) for Cohort 1A animals was conducted on PND111 (males / females) (09-15 June 2017)
Necropsy for recovery animals (Cohort 1B) was conducted on PND141 (males / females) (10 July 2017)

HISTOPATHOLOGY: Yes (see Report, Appendix 1 (Pathology Report)

ORGAN WEIGHTS: Yes (Report, Table 26/27)
Statistics:
The statistical evaluation of appropriate data was performed with the statistical program package of SAS 9.2 (when using Provantis).
In case of the SAS 9.2 software package (within the validated Provantis system) the following decision tree was applied automatically for statistical evaluation of continuous numeric data:
The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.

If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control.
 

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item treatment-related clinical signs were detected for any animals of cohort 1A (used for the 90-day repeated dose toxicity study). Minor clinical signs were recorded for some animals as follows. Red discharge was noted for one Control male (near the snout in the period of PND 98-119) and one Mid-dose male (near the left eye in the period of PND 84-119). Hyperactivity was recorded for a Low-dose female in the periods of PND 29-121.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no statistically significant, test item-related effect on the Low-dose F1A-animals. There was a slightly significant decrease (about 7%, p<0.05) in the body weight of the High-dose F1A-males (at PND120), while the terminal body-weight gains (PND21-120) remained insignificant for all male groups. On the other hand, the terminal mean body weights of females of the Mid- and High-dose group were statistically lower than those of the controls (-9 and -18 %, respectively), more marked for the mean body-weight gains from PND21 to PND120 (-10% and -20%, respectively) (see Report Table 20). The significant decreases in mean body weight in the male and female High-dose groups are considered to be treatment-related.

Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test item-related reduced food consumption was observed for High-dose females of Cohort 1A (minus 10.6% after PND120, p<0.01), but no similar effect was observed in High-dose males, or in the Mid- and Low-dose groups of either sex (see Report, Table 21).

Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No test item-related changes were noted at ophthalmoscopy examination. All examined animals were found to be normal even at early time-point (PND 36-42) as well as before termination (PND 101-110).
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
When compared to the controls, there were no differences in any haematological parameters that could be considered toxicologically significant in any test item-treated group of Cohort-1A males and females, although statistically significant in some cases: decreased mean cell haemoglobin and MCC (p<0.05 or p<0.01) in the Mid- and High-dose males, increased platelet count (p<0.05) in High-dose females, and decreased relative amount of reticulocytes in the Mid- and High-dose females (p<0.05 and p<0.01, respectively) (see Report, Table 24, and Appendix 4.4.3). However, all those values did not suggest a dose response, while the same time were within the normal physiological and historical control range.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes on the serum chemistry parameters of the animals in Cohort 1A, although certain parameters showed statistically significant differences when compared to the controls: decreased cholesterol concentration in the Mid- and High-dose males (p<0.01), decreased albumin concentration and albumin/globulin ratio in High-dose females (p<0.05 or <0.01, respectively) (see Report, Table 25, and Appendix 4.4.3).

Urinalysis findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in the test item-exposed groups were observed in the urinalysis of Cohort 1A, concerning urine volume, pH or specific gravity, urinary constituents and appearance (see Report, Appendix 4.4.3).

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no test item-related effects in the neurological assessment, as there were no observed differences in animal behaviour, general physical condition (grip strength, locomotor activity) or in the reactions to different types of stimuli in the control or test item-treated groups (see Report, Table 22, and Appendix 4.1.5).

Immunological findings:
no effects observed
Description (incidence and severity):
In an immunophenotyping analysis on splenic lymphocyte samples of Cohort 1A to determine the relative and absolute counts of six different lymphocyte subpopulations, there were no test item-related shifts in the immunological steady-state distribution of "helper" (CD4+) or cytotoxic (CD8+) thymus-derived lymphocytes or natural killer (NK) cells in males or females (see Report, Table 30/31, and Appendix 10).
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant increases, considered test item-related, were present in the liver weights in both sexes of the High-dose group and in the Mid-dose males (see Report, Table 27/28). These differences were supported by test item-related histopathological findings.
Other statistical differences were found in High-dose males: brain, kidney and thyroid (only when adjusted to body weight), and in High-dose females: brain, adrenals, thyroid, uterus (adjusted to body weight), heart, kidney, spleen and ovary. But based on microscopic examination these organ-weight differences were considered as secondary changes due to the body weight effect, without evidence of a direct effect of the test item. For further information: see `Details on results´.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A:
Treatment-related changes were recorded in the liver of High-dose males. No treatment-related changes were noted in the High-dose females. Pale mottled/diffuse discoloration was seen in 4/22 males of the High-dose only (a similar incidence of this finding as in the parental (F0) generation animals). All other changes were incidental, a common background or related to the oestrous cycle.

Cohort 1B:
At the terminal necropsy, a total of 2/18 High-dose males had hepatic changes, pale and enlarged liver in 1/18 and pale liver in 1/18 males with liver weights were slightly increased in the High-dose only. All other changes were considered to be incidental or common background findings. In recovery subset animals, no significant liver-weight or any hepatic effects and no other no treatment-related changes were detected at necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A:
Treatment-related histopathological findings were noted in examined High-dose animals.
There was a mixed form of hepatocellular vacuolation observed in the livers of High-dose males and females: centrilobular (mainly) and periportal zonation were seen. The severity varied from minimal to moderate with frequency of 19/22 males and 13/24 females. A severe intensity with diffuse distribution altered liver was found in 1/22 males. No similar vacuolation was recorded in controls. This observation was considered to be non-adverse. In addition to the standard histopathology examination, for F1 parental females only, a quantitative assessment of primordial follicles and corpora lutea was conducted. There were no meaningful differences in total number (left and right ovaries) of primordial follicles and corpora lutea between Control and High-Dose females

Cohort 1B:
High-dose recovery animals were examined (The 1A cohort provided the histopathology evaluation for the F1 animals). No treatment-related microscopic changes were observed in examined males and females.
Hepatocellular vacuolation of the liver was observed in 2/5 males exposed at a dietary concentration of 1500 mg/kg diet; however, macrovesicular forms were considered to be common background observations similar to those commonly seen in control animals. Hence, it was considered there was no effect in these recovery animals.
In addition, a quantitative assessment of primordial follicles and corpora lutea was conducted in the females according to the Study plan. There were no meaningful differences in total number (left and right ovaries) of primordial follicles and corpora lutea between Control and High-dose females.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Cohort 1A
Additional parameters (balanopreputial separation for males, vaginal opening and oestrus cycle for females) were also examined to follow the sexual development of Cohort 1A animals.
Balanopreputial separation: A slightly slower but not statistically significant development in the High-dose males was observed in the Cohort 1A (retardation of about 1-2 days (PND42 vs. PND40 to 41) (see Report, Figure 15, and Appendix 3.9). However, since the body weight at the time of balanopreputial separation was significantly lower than controls, the slower development was considered to be secondary to lower body weight and not a direct test item effect.

Vaginal patency: Vaginal patency (vaginal opening) was normal for all female pups beginning on PND 22. The body weight of each female was recorded on the day of vaginal patency. No effect on this parameter was observed in the Low-, Mid- and High-dose groups of Cohort 1A females (see Report, Figure 16, Table 23, and Appendix 3.9).

Oestrus cycle: The Oestrous cycles did not show significant differences between the groups.

Sperm Analysis: Sperm motility and morphology as well as number of sperms were examined in the study for Cohort 1A. No treatment-related effect was seen in any dose groups of this cohort (see Report, Table 29, and Appendix 4.6.3).

Thyroid hormone analysis: No toxicologically relevant changes in the test item exposed groups were observed in the thyroid hormone values of Cohort 1A (see Report, Table 26, and Appendix 4.4.3).
A statistically higher value (p<0.05) was recorded for the High-dose females for T4 (of 45.45 pg/mL), however the Parental T4 range (47.9 - 60.8 pg/mL for animals of approximately the same age) shows that the control mean for the 1A group is relatively low. The High-dose female 1A data is not higher than the normal range, hence the statistical difference is not considered to be of biological significance.

Cohort 1B
Cohort 1B animals were dedicated to obtaining additional histopathology data in cases of suspected reproductive or endocrine toxicants, or when results from Cohort 1A were equivocal. No such an assessment was deemed necessary in the study. Hence, the data from in-life and at clinical pathology and necropsy were tabulated and included in the Appendices attached to the study report but are not reported, since the requirement for this additional data was not triggered. The macroscopic data from necropsy has been provided to supplement the findings in the 1A cohort. Additionally, the histopathology data of the recovery animals of the 1B cohort has also been provided.

The following information for Cohort 1B was collected and/or parameters measured during the in life phase: clinical observations, body weight and body weight gain, food consumption, clinical pathology (haematology, clinical chemistry, urinary analysis, necropsy findings, organ weights (absolute and adjusted to body / brain weight) and histopathology. All the results are provided in the appropriate appendices attached to the study report, but no formal evaluations were made, as it was not necessary based on the Cohort 1A results.
Details on results:
COHORT 1A:
CLINICAL CHEMISTRY:
There were no test item-related changes on the serum chemistry parameters of the animals in Cohort 1A, although certain parameters showed statistically significant differences when compared to the controls: decreased cholesterol concentration in the Mid- and High-dose males (p<0.01), decreased albumin concentration and albumin/globulin ratio in High-dose females (p<0.05 or <0.01, respectively).

Cholesterol: A similar decrease but without statistical significance was seen in the High-dose females (see Report, Table 25, and Appendix 4.4.3). However, the observed mean values were within the normal physiological range (mean of historical control ± 2-times standard deviation, SD) in all cases (males and females). Furthermore, the control mean was at the maximum of the historical range, while the High-dose was in the middle of the historical range. Hence, from this data alone, it would have been considered to be unrelated to treatment. In this study in the P0 generation, the control values were also relatively high, and there were slightly but statistically significantly (p<0.05) lower cholesterol values, hence a relationship with treatment cannot be totally excluded, but the High-dose group values were in the middle of the historical normal range. The differences in the High-dose males in 1A-cohort are considered to be of equivocal relationship with treatment, but in the absence of any other associated findings, when the values were considered completely normal, were considered not to reflect an adverse change .

Protein: Decreased values for parameters related to protein content (total protein concentration, albumin concentration and albumin/globulin ratio) were observed in High-dose females, the difference from control was statistically significant for albumin and A/G ratio (p<0.05 or <0.01, respectively). However, no similar trends were seen in High-dose males (actually in High-dose males an opposite change, statistically significant increase (p<0.05) in total protein content was detected). Furthermore, all the observed values were within the normal physiological range (mean of historical control ± 2SD). Therefore, the observed differences between values were considered as having no biological relevance (the actual values of female control animals were higher than usual based on the historical control database) and not being related to test item administration.

Ions: Statistically significant changes (at p<0.05 level) were recorded for the concentrations of some ions: increased sodium and calcium concentration were noted for High-dose males, and increased chloride concentration was noted in High-dose females when compared to control animals. However, the differences were minor (less than 5%), no similar trends were seen in the other sex in any of those cases. Furthermore, all those data were within the normal physiological control and/or historical control range. Therefore, they were considered as animal variability, not related to the test item.


BODY WEIGHT AND WEIGHT CHANGES:
There was no test item related effect on the Low dose group animals. There was no effect on body weight of males at any dose level. Females in the Mid- and High-dose groups had a lower starting body weight at the High dose (and to some extent at the Mid dose) and they had a lower growth rate compared to controls at the Mid and High dose level.

Although statistically significant decrease (by 7%, p<0.05) was observed in the body weight at the end of the evaluation period (PND 120) for High-dose males when compared to the control, this was not considered as clear evidence of test item effect, as the starting body weight at weaning (on PND 21) also showed a similar difference (approx. 8%, p<0.05). As no significant difference was seen in the body weight gain of the High-dose males during the PND 21-120 period, the observed values were considered as showing no test item-related effect. Neither were there significant changes of body weight in the Mid- and Low-dose groups.

However, in case of Cohort 1A females, the original difference in the starting body weight of Mid- and High dose animals compared to control (by 4.9% and 9.0%) became wider by the end of the evaluation period (9.0% in case of Mid-dose females and 18.1% in case of High dose females, both significant at p<0.01). Furthermore, the body weight gain during this period (PND 21-120) was statistically significant in the Mid- and High-dose females (10.3% and 20.3%, p<0.01 in both cases) compared to the control group. These values indicated a test item-related effect in the females of the Mid-and High-dose group, although the impact of a lower starting body weight is difficult to evaluate. There were no significant changes in the Low dose females of this cohort.

ORGAN WEIGHT:
Terminal body weights of males and females were statistically significantly lower in the terminal body weights (by 9.0%, p<0.01) was also observed in Mid-dose females. No changes in terminal body weights were observed in Mid-dose males or Low-dose animals when compared to the controls.

Statistically significant differences, considered test item-related, were present in the liver weights in both sexes of the High-dose group. These differences were supported by test item-related histopathological findings. There was an increase in absolute and relative (to body and brain) liver weights in the High-dose males, and for body weight-adjusted liver weights in the High-dose females. In the High-dose males, absolute liver weights were statistically significantly higher by 20.1% (p<0.01), body-related by 29.6% (p < 0.01), and by 19.9% (p < 0.01) when brain-related. In High-dose females, liver weight-adjusted body were increased by 15.0% (p < 0.01) but values were slightly lower than control for absolute and brain-adjusted weights. A statistically significant increase considered test item-related was defined in the liver of Mid-dose males. When related to body and to brain weights, liver weights were larger by 11.5% (p<0.01) and by 10.8% (p <0.05), respectively. No statistically significant differences were reported in Mid-dose female livers.

In both sexes of the High-dose group, the treatment-related lower body weight had a consequence with some statistically significant lower organ weights. But based on microscopic examination these organ weight differences were considered as secondary changes due to the body-weight effect, without evidence of a direct effect of the test item. These statistical differences were seen in High-dose males concerning brain, kidney and thyroid (only when adjusted to body weight), and in High dose females concerning brain, adrenals, thyroid, uterus (adjusted to body weight), heart, kidney, spleen and ovary.

There were no significant differences considered to be treatment-related in the Low-dose group (males and females) compared to controls.

GROSS PATHOLOGY:
Treatment-related changes were recorded in the liver of High dose males. No treatment-related changes were noted in the High dose females.

Pale mottled/diffuse discoloration was seen in 4/22 males of the High-dose only (a similar incidence of this finding as in the parental (F0) generation animals). All other changes were incidental, a common background or related to the oestrous cycle.

HISTOPATHOLOGY: see Results of Examination

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
42 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 7. Selected body weight parameters of Cohort 1A animals

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated males

20

23

21

22

 

Male, Body weight on PND21 (g)

68.7

67.5

64.9

63.1**

D

difference (%)

-1.8

-5.5

-8.2

 

Male, Body weight on PND120 (g)

595.3

610.3

584.6

555.3*

DN

difference (%)

2.5

-1.8

-6.7

 

Male, Body weight gain PND21-120 (g)

526.6

542.8

519.7

492.2

NS

difference (%)

3.1

-1.3

-6.5

 

Number of evaluated females

22

23

21

24

 

Female, Body weight on PND21 (g)

65.8

62.9

62.6

59.9**

D

difference (%)

-4.5

-4.9

-9.0

 

Female, Body weight on PND120 (g)

331.3

312.9

301.5**

271.4**

DN

difference (%)

-5.6

-9.0

-18.1

 

Female, Body weight gain PND21-120 (g)

265.5

250.0

238.9**

211.5**

DN

difference (%)

-5.8

-10.0

-20.3

 

Notes: Data (group mean values) were rounded to one decimal place.

PND = post-natal day

Statistical significance compared to control: *: atp<0.05, **: at p<0.01

D: Dunn test; DN: Dunnett’s Multiple Range Test, NS: Statistically not significant when comparedto control

Table 8. Selected food consumption parameters of Cohort 1A

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated males

20

24

21

22

 

Male, Food consumption (PND21-120) (g/day)

26.88

28.64

27.78

26.78

NS

difference (%)

6.6

3.4

-0.4

 

Number of evaluated females

22

23

21

24

 

Female, Food consumption (PND21-120) (g/day)

19.43

19.40

19.01

17.37**

DU

difference (%)

-0.2

-2.2

-10.6

 

Notes: Data (group mean values of daily food consumption) were rounded to two decimal places.

PND = post-natal day

Statistical significance compared to control: *: at p<0.05, **: at p<0.01

DU: Dunn test, NS: Statistically not significant compared to control

Table 9. Selected FOB and SMART parameters of Cohort 1A

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated male animals

20

23

21

22

 

Males, Landing foot splay (hind paws)

90.0

78.23

83.70

76.41

NS

Males, Grip-strength (forelimbs)

1809.2

1813.3

1783.85

1768.9

NS

Males, Grip-strength (hind limbs)

453.3

477.4

436.05

454.1

NS

Males, Total travelled distance (cm)

7992.6

8767.8

8070.167

8632.9

NS

Number of evaluated female animals

22

23

21

23

 

Females, Landing foot splay (hind paws)

75.5

65.7

67.2

60.2*

DN

Females, Grip-strength (forelimbs)

1321.0

1269.5

1274.5

1275.0

NS

Females, Grip-strength (hind limbs)

344.0

318.1

329.2

294.3

NS

Females, Total travelled distance (cm)

9523.8

11426.3

10894.8

10352.0

NS

Notes: Data (group mean values) are rounded to one decimal place.

Statistical significance compared to control: *: at p<0.05, **: at p<0.01

DU: Dunn Test, DN: Dunnett’s Multiple Range Test, NS: Statistically not significant compared to control

Table 10. Summary of selected haematology parameters (Cohort 1A parental animals)

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Male, Mean Cell Haemoglobin (pg)

18.06

17.35*

17.30*

17.22**

DU

difference (%)

-3.9

-4.2

-4.6

 

Male, MCHC (g/dL)

33.88

33.05

32.76**

32.63**

DU

difference (%)

-2.4

-3.3

-3.7

 

Male, Platelet count (K/μL)

965.8

995.6

990.5

984.8

NS

difference (%)

3.1

2.6

2.0

 

Male, Relative amount of reticulocytes (%)

2.420

2.409

2.471

2.382

NS

difference (%)

-0.5

2.1

-1.6

 

Female, Mean Cell Haemoglobin (pg)

18.63

18.49

18.40

18.10

NS

difference (%)

-0.8

-1.3

-2.9

 

Female, MCHC (g/dL)

33.27

33.58

33.15

33.08

NS

difference (%)

0.9

-0.4

-0.6

 

Female, Platelet count (K/μL)

862.6

895.5

965.2

959.0*

DN

difference (%)

3.8

11.9

11.2

 

Female, Relative amount of reticulocytes (%)

2.60

2.42

2.30*

2.033**

DU

difference (%)

-7.0

-11.4

-21.8

 

Notes: Data (group mean values, n=20-24) were rounded as appropriate (to 1-3 decimal places).

Statistical significance compared to control: *: at p<0.05, **: at p<0.01

DU: Dunn Test, DN: Dunnett’s Multiple Range Test, NS: Statistically not significant compared to control

Table 11. Selected clinical chemistry parameters of Cohort 1A animals

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Male, Cholesterol (mmol/L)

2.190

1.930

1.758**

1.669**

DN

difference (%)

-11.8

-19.7

-23.8

 

Male, Total protein (g/L)

57.92

58.19

58.90

60.18*

DN

difference (%)

0.5

1.7

3.9

 

Male, Albumin (g/L)

31.59

31.77

32.12

33.01

NS

difference (%)

0.6

1.7

4.5

 

Male, A/G ratio

1.20

1.20

1.20

1.22

NS

difference (%)

-0.4

0.4

1.9

 

Male, ALKP (U/L)

102.7

92.8

90.5

105.6

NS

difference (%)

-9.6

-11.8

2.9

 

Male, ALT / GPT (U/L)

43.5

47.9

52.8

52.9*

DU

difference (%)

10.0

21.3

21.6

 

Male, Bile acid (μmol/L)

12.770

12.867

14.974

16.295*

DU

difference (%)

0.8

17.3

27.6

 

Female, Cholesterol (mmol/L)

2.408

2.117

2.111

1.943

NS

difference (%)

-12.1

-12.3

-19.3

 

Female, Total protein (g/L)

67.05

64.49

65.93

64.51

NS

difference (%)

-3.8

-1.7

-3.8

 

Female, Albumin (g/L)

41.50

39.35

40.05

37.99*

DN

difference (%)

-5.2

-3.5

-8.5

 

Female, A/G ratio

1.636

1.565

1.548

1.442**

DN

difference (%)

-4.3

-5.4

-11.9

 

Female, ALKP (U/L)

52.9

50.6

62.6

73.4**

DU

difference (%)

-4.3

18.4

38.7

 

Female, ALT / GPT (U/L)

46.9

52.6

55.5

50.8

NS

difference (%)

12.2

18.5

8.3

 

Female, Bile acid (μmol/L)

14.050

16.408

16.636

17.875*

DU

difference (%)

16.8

18.4

27.2

 

Notes: Data (group mean values, n=20-22) were rounded as appropriate (to 1-3 decimal places).

ALT (GPR): Alanine Aminotransferase (Glutamate-pyruvate transaminase); ALKP: Alkaline phosphatase

Statistical significance compared to control: *: at p<0.05, **: at p<0.01

DN: Dunnett’s Multiple Range Test; DU: Dunn test; NS: Statistically not significant when compared to control

Table 12. Organ weight data of Cohort 1A males

 

Organ weight

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated animals

20

23

21

22

 

Terminal body weight, g

571.2

584.9

560.1

530.1*

DN

(difference %)

2.4

-1.9

-7.2

 

Liver (absolute), g

15.824

16.906

17.300

19.003**

DN

(difference %)

6.8

9.3

20.1

 

Liver (relative to body), %

2.770

2.889

3.089**

3.589**

DU

(difference %)

4.3

11.5

29.6

 

Liver (relative to brain), %

692.08

739.60

767.05*

829.84**

DN

(difference %)

6.9

10.8

19.9

 

Notes: Organ weight data (group mean values) were rounded to two or three decimal places.

Statistical significance compared to control: *: atp<0.05, **: at p<0.01

DN: Dunnett’s Multiple Range Test, DU: Dunn’s test, NS: Statistically not significant compared to control

 

Table 13. Organ weight data of Cohort1 1A females

 

Organ weight

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated animals

22

23

21

22

 

Terminal body weight, g

317.1

298.4

288.6**

257.7**

DN

(difference %)

-5.9

-9.0

-18.8

 

Liver (absolute), g

10.223

9.906

9.680

9.570

NS

(difference %)

-3.1

-5.3

-6.4

 

Liver (relative to body), %

3.234

3.328

3.365

3.717**

DU

(difference %)

2.9

4.1

15.0

 

Liver (relative to brain), %

497.73

481.71

473.12

471.77

NS

(difference %)

-3.2

-4.9

-5.2

 

Notes: Organ weight data (group mean values) were rounded to two or three decimal places.

Statistical significance compared to control: *: atp<0.05, **: at p<0.01

DN: Dunnett’s Multiple Range Test, DU: Dunn’s test, NS: Statistically not significant compared to control

Table 14. Immunophenotyping of Cohort 1A (absolute counts)

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Males

 

Number of analysed samples

20

21

21

22

NS

Number of splenocytes (cell/μL)

206.9

178.6

241.0

240.9

NS

Number of B cells (cell/μL)

84.2

75.5

103.8

98.6

NS

Number of NK cells (cell/μL)

8.6

7.6

7.5

10.5

NS

Number of NKT cells (cell/μL)

11.0

9.1

13.8

11.0

NS

Number of T cells (cell/μL)

79.8

64.6

85.0

86.4

NS

Number of Helper T cells (cell/μL)

54.4

42.5

61.0

56.2

NS

Number of Cytotoxic T cells (cell/μL)

18.8

14.8

20.0

21.3

NS

Females

 

Number of analysed samples

22

21

21

24

NS

Number of splenocytes (cell/μL)

203.9

176.7

228.7

237.8

NS

Number of B cells (cell/μL)

83.6

77.0

96.2

102.5

NS

Number of NK cells (cell/μL)

8.4

6.5

8.0

8.5

NS

Number of NKT cells (cell/μL)

10.5

8.5

13.3

9.6

NS

Number of T cells (cell/μL)

75.0

66.2

83.9

87.1

NS

Number of Helper T cells (cell/μL)

50.6

45.5

61.1

59.1

NS

Number of Cytotoxic T cells (cell/μL)

17.3

14.6

18.5

20.1

NS

Notes: Data (group mean values) are rounded to one decimal place.

NS: statistically not significant, according to the decision tree (relevant phase report), where Dunnett’s test is used for significance.

 

Table 15. Immunophenotyping of Cohort 1A (relative counts)

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Males

 

Number of analysed samples

20

21

22

22

NS

Number of B cells (cell/μL)

40.5

41.1

42.6

40.2

NS

Number of NK cells (cell/μL)

4.2

4.4

3.3

5.3

NS

Number of NKT cells (cell/μL)

5.4

5.2

6.6

4.9

NS

Number of T cells (cell/μL)

39.0

36.9

35.5

36.5

NS

Number of Helper T cells (cell/μL)

68.2

66.2

91.8

65.4

NS

Number of Cytotoxic T cells (cell/μL)

22.9

22.4

32.9

23.9

NS

Females

 

Number of analysed samples

22

21

21

24

NS

Number of B cells (cell/μL)

40.2

42.7

41.6

42.8

NS

Number of NK cells (cell/μL)

4.3

3.7

3.7

3.8

NS

Number of NKT cells (cell/μL)

5.2

4.9

6.5

4.2

KW*

Number of T cells (cell/μL)

37.5

38.2

36.6

36.9

NS

Number of Helper T cells (cell/μL)

66.8

68.3

89.7

67.7

NS

Number of Cytotoxic T cells (cell/μL)

23.2

22.3

27.5

23.2

NS

Notes: Data (group mean values) are rounded to one decimal place.

NS: statistically not significant, according to the decision tree (relevant phase report), Dunnett’s test for significance.

Table 16. Summary of thyroid hormone analysis (Cohort 1A)

 

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Male, T4 (ng/mL)

56.25

51.91

51.97

56.20

NS

difference (%)

-7.7

-7.6

-0.1

 

Male, TSH (pg/mL)

1847.8

1689.7

2169.1

2414.1

NS

difference (%)

-8.6

17.4

30.6

 

Male, Terminal body weight (g)

571.2

584.9

560.1

530.1*

DN

difference (%)

2.4

-1.9

-7.2

 

Male, Thyroid & parathyroid weight (g)

0.0322

0.0345

0.0325

0.0347

NS

difference (%)

7.5

1.2

7.9

 

Female, T4 (ng/mL)

33.97

38.89

33.45

45.45*

DN

difference (%)

14.5

-1.5

33.8

 

Female, TSH (pg/mL)

2118.7

1505.0

1441.9

2606.9

NS

difference (%)

-29.0

-31.9

23.0

 

Female, Terminal body weight (g)

317.1

298.4

288.6**

257.7**

DN

difference (%)

-5.9

-9.0

-18.8

 

Female, Thyroid & parathyroid weight (g)

0.0250

0.0259

0.0235

0.0242

NS

difference (%)

3.6

-6.1

-3.1

 

Notes: Data (group mean values, n=10) were rounded to one to four decimal places

Applicant's summary and conclusion

Conclusions:
Based on the effects observed in Cohort 1A and Cohort 1B of this study, the NOAEL for systemic toxicity was determined to be 42 mg/kg bw/day, corresponding to 500 mg OAPP/kg diet (nominal). Additionally, exposure to the test material did not result in any immunotoxic effects. The effect was more marked in the treated females.
Executive summary:

A key combined extended one-generation reproductive toxicity (EOGRTS) / sub-chronic oral toxicity study was conducted according to Guideline recommendations (OECD 443 / OECD 408) to evaluate specific life stages not covered by other types of toxicity studies and to test for effects that may occur as a result of pre- and post-natal chemical exposure to the test material (Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol (OAPP), EC# 700 -960 -7). The OECD 443 study was combined with the OECD 408 (90-day rat) study using the F1 generation (hence exposure was in utero to weaning, then effectively further 100 days), to evaluate sub-chronic toxicity of the test material.

 

The test material was administered continuously in the diet to selected pups (male and female Wistar rats) of the F1 generation (Cohort 1A: at least 20 animals/sex/group) at nominal concentrations in the diet of 0, 150, 500, or 1500 ppm [mg/kg diet] for 100 days after weaning, i.e. from PND21 through PND120. This part of the study also included 30-day recovery animals (Cohort 1B: 5 males and 5 females in the Control and High-dose groups, respectively).

 

There were no mortalities after weaning, no adverse clinical signs, and no effects were observed on the neurotoxicity parameters evaluated.

 There was no statistically significant, test item-related effect on the Low-dose F1A-animals. There was a slightly significant decrease (about 7%, p<0.05) in the body weight of the High-dose F1A-males (at PND120), while the terminal body-weight gains (PND21-120) remained insignificant for all male groups. On the other hand, the terminal mean body weights of females of the Mid- and High-dose group were statistically lower than those of the controls (-9 and -18 %, respectively), more marked for the mean body-weight gains (-10% and -20%, respectively (from PND21-120) (see Report Table 20). The decreases in mean body weight in the male and female High-dose groups are considered to be treatment-related.

 

No adverse treatment-related effects on haematology, clinical chemistry, or urine analysis were observed in any dose group. All examined animals were found to be normal during the ophthalmoscopy examinations.

 

Reductions in some organ weights were observed, but considered to be a consequence of lower body weights, and not an adverse effect.

A treatment-related mixed form of hepatocellular vacuolation occurred in the High-dose males and females, visualised as pale lobes seen in some males at necropsy. A statistically significant liver-weight increase, considered treatment-related, was present in both sexes in the High-dose, and to a slight extent in Mid-dose males. Centrilobular (mainly) and periportal zonation were seen at histopathology; similar to the parental animals, the severity varied mainly from minimal to moderate, and there were proportional frequencies viewed in the male and females. The hepatic morphological changes were considered to be an adaptive, non-adverse response of the liver.

 

In the recovery animals (high-dose vs. control groups), there were no significant differences in liver weights and no treatment-related macroscopic or microscopic hepatic changes in examined males and females after the recovery period. This confirms the adaptive, non-adverse nature of the terminal hepatic observations. Additionally, a quantitative assessment of primordial follicles and corpora lutea conducted in terminal and recovery females did not show any meaningful differences in total number of primordial follicles and corpora lutea between Control and High-dose females.

 

Based on the effects observed in Cohort 1A and Cohort 1B of this study, the NOAEL for systemic toxicity was determined to be 42 mg/kg bw/d, administered to rats in the food at a level of 500 mg/kg diet (nominal). Additionally, exposure to the test material did not result in any immunotoxic effects.