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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes
Limit test:
yes
Justification for study design:
Decision number: CCH-D-2114482123-55-01/F
Based on Article 41 of Regulation (EC) No 1907/2006 (the REACH Regulation), ECHA requests you to submit information on:
Extended one-generation reproductive toxicity study (Annex X), Section 8.7.3.; test method: EU B.56/OECD TG 443) in rats, oral route, with the registered substance, specified as follows:
- At least two weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) with extension to mate the Cohort 1B animals to produce the F2 generation.

Test material

Constituent 1
Chemical structure
Reference substance name:
Propyl acetate
EC Number:
203-686-1
EC Name:
Propyl acetate
Cas Number:
109-60-4
Molecular formula:
C5H10O2
IUPAC Name:
propyl acetate
Test material form:
liquid
Details on test material:
TEST SUBSTANCE: propyl acetate
Specific details on test material used for the study:
Identity n-Propyl acetate
Batch nos. P1118118AA, 50000097311
CAS no. 109-60-4
Expiry date 31 January 2021, 31 December 2021
Appearance Colourless liquid
Storage conditions Room temperature
ERBC nos. 16776, 17261

The determination of the identity, strength, purity, composition and stability of the test item was the responsibility of the Sponsor. The Sponsor declared that the characterisation of the test item was carried out according to GLP. A sample of test item from
each batch was taken and will be stored in the archives of ERBC for 10 years prior to disposal.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar Hannover rat was the species and strain of choice, as it is accepted by many regulatory authorities for reproductive toxicology studies and there are ample experience and background data on this species and strain.
The test item was administered orally, by gavage. The oral route was selected as it is a possible route of exposure of the test item in man.
Dose levels were selected by the Sponsor based on information from a preliminary study (ERBC non-GLP Study No. Y0490).
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, CRL, Sain Nuelles, France.
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: (P) 8 wks; (F1) 8 wks
- Weight at study initiation: (P) Males: 214-256 g; Females: 210-229 g;
- Fasting period before study: fasting procedure was only necessary for clinical pathology/haematological investigations
- Housing:
P
From arrival to pairing, the animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week. During mating, animals were housed one male to one female in clear polysulfone cages measuring approximately 42.5×26.6×18.5cm with a stainless steel mesh lid and floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed as necessary. After mating the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Suitable nesting material was provided inside suitable bedding bags and changed as necessary. Additional nesting material (Scobis 0 Mucedola) was provided as necessary.

Cohorts 1A, 1B
The animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During mating of Cohort 1B, animals were housed one male to one female in clear polysulfone cages measuring approximately 42.5×26.6×18.5cm with a stainless steel mesh lid and floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed as necessary.
After mating the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0Mucedola) was provided as necessary. This material was changed as necessary (at least 2 times a week).
- Diet: ad libitum
- Water: ad libitum (except when urinalysis was performed)
- Acclimation period: at least two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 2
- Humidity (%): 55 +- 15
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Vehicle
The vehicle was corn oil.

Preparation of test item
The required amount of n-Propyl acetate was dissolved in the vehicle. The preparations were made daily unless specified otherwise (concentrations of 25, 75 and 250mg/mL) and delivered at room temperature protected from light. Concentrations were calculated and expressed in terms of test item as supplied.

Administration of test item
The test item was administered orally by gavage at a dose volume of 4mL/kg body weight.
Control animals received the vehicle alone at the same dose volume.
The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: [vaginal plug or sperm in vaginal smear] referred to as [day 0] post coitum
- If mating did not occur after 14 days of cohabitation the animals were separated.
- After successful mating each pregnant female was caged (how): The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm).

Mating-P
Pairing was monogamous (one male to one female). Each female of the Parental generation was placed in the home cage of a male from the same group randomly selected and left overnight.
Vaginal smears were taken from the day after the start of pairing, in the morning, during pairing up to the day of positive identification of mating (spermidentification, vaginal plug in situ or copulation plugs found in the cage tray).
The dates of pairing, of insemination and of parturition were recorded and the pre-coital interval (pairing to insemination) and the duration of pregnancy (insemination to parturition) were calculated.

Mating-1B
Pairing was monogamous (one male to one female). Exceptions can arise in the case of occasional deaths of males. Mating started at least on 13weeks of age. Brother-sister pairing and other closely related pairings were avoided. Each female was placed in the home cage of the male, randomly selected (males) from the same group and left overnight. Vaginal smears were taken from the day after the start of pairing, in the morning, during pairing up to the day of positive identification of copulation (spermidentification or vaginal plug in situ. If mating did not occur after 14 days of cohabitation the animals were separated. The
female was killed at least 25 days after the last mating session.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis has been performed in a separate study (ERBC Study no. A3871) in order to validate the analytical method and the preparation procedure. The stability of the preparations has also been verified in the same study. Stability was verified at time points of 28 hours at room temperature and after 8 days at room temperature in the concentration range of 10 to 250 mg/mL. The software used was Chromeleon 7, version 7.2.9 (Thermo Scientific).
Samples of the preparations prepared during the current study (the first and the last week of treatment of parental generation and F1 generation (Cohorts 1A and 1B) were analysed to check the concentration. Chemical analysis were carried out by the Analytical Chemistry Department at ERBC. The software used for this activity was Chromeleon 7, version 7.2.9 (Thermo Scientific).
Duration of treatment / exposure:
P/F0
Males
Males were treated once a day for at least 2 weeks prior to pairing and during mating up to the day before sacrifice (at least 70 days).
Dose volumes were calculated according to individual body weight at weekly intervals.

Females
Females were treated once a day at least 2 weeks prior to pairing, during mating, gestation and post partum periods until the day before sacrifice (after treatment for at least 10 weeks).
Dose volumes were calculated according to individual body weight at weekly intervals up to positive identification of mating and then according to body weight on Days 0, 7, 14 and 20 post coitum and on Days 1, 4, 7, 14 and 21 post partum.

Cohort 1A
Males and females were treated once a day starting from Day 21 of age (Day 21 post partum) up to the day before necropsy for at least 10 weeks.

Cohort 1B
Males
Males were treated once a day starting from Day 21 of age (Day 21 post partum) for at least 10 consecutive weeks prior to pairing and during pairing up to the day before sacrifice (after the weaning of the majority F2 litters). Dose volumes were calculated according to individual body weight at weekly intervals.
Females
Females were treated once a day starting from Day 21 of age (Day 21 post partum) for at least 10 consecutive weeks prior to pairing, during mating, gestation and post partum periods until Day 21 post partum or the day before sacrifice. Dose volumes were calculated according to individual body weight at weekly intervals up to positive identification of mating and then according to body weight on Days 0, 7, 14 and 20 post coitum and on Days 1, 7 and 14 post partum. Dose volumes after Day 14 post partum remained constant.
Frequency of treatment:
Once daily
Details on study schedule:
- F1 parental animals not mated until at least 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: >= 13 weeks (Cohort 1B, 21 days plus 72-77 days of treatment before mating)
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
28 (P/F0) 20 (Cohort 1A), 25 (Cohort 1B)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on information from a preliminary study (ERBC non- GLP Study No. Y0490).
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: yes
Positive control:
N/A

Examinations

Parental animals: Observations and examinations:
Mortality
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post mortem examinations to be carried out during the working period of that day. A complete necropsy was performed in all cases.

Clinical signs
P
All clinical signs were recorded for individual animals. Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.
1A
All clinical signs were recorded for individual animals of Cohort 1A daily from selection.
Starting from Day 21 of age until sacrifice, each animal was observed at least once daily and any clinical signs recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.
Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.
1B
All clinical signs were recorded for individual animals starting from the day of selection (Day 21 post partum) until sacrifice. Each animal was observed at least once daily and any clinical signs was recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

Detailed clinical observations (Home Cage & Open Field) - P
Once before commencement of treatment and at least once a week from the start of treatment until termination, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena (for at least 1 minute). Signs recorded included, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture, response to handling, as well as the presence of clonic or tonic movements, stereotypy (e.g. excessive
grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. All observations were recorded for individual animals.

Detailed clinical observations (Home Cage & Open Field) - Cohort 1A
Starting from Day 21 or Day 22 of age and at least weekly intervals until termination, each animal was examined for detailed clinical examinations. Each animal was removed from the home cage and observed in an open arena (for at least 1 minute).
Signs noted included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture, response to handling, as well as the presence of clonic or tonic movements, stereotypy (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. All observations were recorded for individual animals.

Detailed clinical observations (Home Cage & Open Field) - Cohort 1B
Starting from Day 21 or Day 22 of age and at least weekly intervals until termination, each animal was examined for detailed clinical observation. Each animal was removed from the home cage and observed in an open arena (for at least 1 minute).
Signs noted included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture, response to handling, as well as the presence of clonic or tonic movements, stereotypy (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. All observations were recorded for individual animals.

Observed parameters in P, 1A; 1B, described by an evaluation scale, are as follows:
Observed parameters Evaluation scale
Removal (from cage) Easy, Difficult, Very difficult
Handling reactivity Normal, Slow,Moderate,Marked
Lachrymation Absent, Slight,Marked
Palpebral closure Absent, Slight,Moderate,Marked
Salivation Absent, Slight,Marked
Piloerection Absent, Present
Rearing Absent, Intervals of number of times (i.e. 1- 3, 4- 7, 8- 10)
Spasms Absent, Tonic spasms, Clonic spasms, Tonic- clonic spasms
Myoclonia Absent, Present
Mobility impairment Absent, Slight,Moderate,Marked
Arousal (animal activity) Very slow, Slow, Normal,Moderate,Marked
Vocalisation Absent, Present
Stereotypies Absent, Present
Unusual respiratory pattern Absent, Present
Bizarre behaviour Absent, Present
Urination Absent, Intervals of number of times (i.e. 1- 3, 4- 6)
Defecation Absent, Intervals of number of times (i.e. 1- 3, 4- 6)
Tremors Absent, Present
Gait (one of the following options) Normal, Ataxia (Slight,Moderate,Marked), Hunched posture (Slight,Moderate, Severe), Pronation, Forelimbs drag (Slight,Moderate,Marked), Hindlimbs drag (Slight,Moderate,Marked)
All observed parameters are reported in a group incidence table.

Body weight
Males-P
Males were weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.
Females-P
Females were weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter until positive identification of mating.
Females were also weighted on Days 0, 7, 14 and 20 post coitum and on Days 1, 4, 7, 14 and 21 post partum.

1A
Animals were weighed on the day of allocation to groups (Day 21 of age) and then at weekly interval until termination and on the day of necropsy.
Body weight was also recorded in Cohort 1A animals on the day when they attained puberty (completion of preputial separation or vaginal patency).

1B
Males
Males were weighed on the day of allocation to groups (Day 21 of age) and then weekly until termination and on the day of necropsy.
Females
Females were weighed on the day of allocation to groups (Day 21 of age) and then weekly until positive identification of mating. After mating body weight was also performed on Days 0, 7, 14 and 20 post coitum and on Days 1, 4, 7, 14 and 21 post partum.
Body weight was also recorded in Cohort 1B animals on the day when they attained puberty (completion of preputial separation or vaginal patency).


Food consumption
Males-P
Food consumption was recorded at weekly intervals starting from Day 1 of dosing and in the same days as body weights (except during cohabitation) up to pairing. In addition, for males of P generation the food consumption was recorded at weekly intervals from the end of the mating period for all males until termination.
Females-P
Food consumption was measured weekly starting from Day 1 of dosing and on the same days as body weights up to mating.
Food consumption of females of P generation was measured on Days 7, 14, and 20 post coitum, starting from Day 0 post coitum and on Days 4, 7, 14 and 21 post partum starting from Day 1 post partum.

1A
Food consumption was measured weekly from Day 21 of age until termination.

1B
Males
Food consumption was recorded at weekly intervals (whenever possible) for males starting from Day 21 and in the same days as body weights (except during cohabitation), up to pairing. In addition, food consumption was recorded at weekly intervals from the end of the mating period until termination.
Females
Food consumption was measured weekly on the same days as body weights up to mating and on Days 7, 14, and 20 post coitum, starting from Day 0 post coitum and on Days 4, 7, 14 and 21 post partum starting from Day 1 post partum.
Oestrous cyclicity (parental animals):
P
The assessment of oestrous cycles, with vaginal smears, was performed once daily from allocation to dosing.
For remaining stock females, oestrous cycle was monitored by vaginal smears for the same period of the allocated females. These data are not tabulated but archived with all raw data.
Vaginal smears were taken in the morning starting from at least 2 week before pairing and throughout the mating period until a positive identification of copulation was made.
The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle;
2. pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Vaginal smears were also taken from all stock females from allocation to period up to the start of dosing females formallocated females and on the day of necropsy.
No vaginal smears were taken in animals sacrificed for humane reasons.

Cohort 1A/1B
Vaginal smears
Female pups
Vaginal smearswere examined daily for all F1 females of Cohort 1A and 1B, after the onset of vaginal patency, until the first cornified smear (first oestrous) was recorded, in order to determine the time interval between these two events.
Adult F1 females
Oestrous cycles for all females of Cohorts 1A and 1B were also monitored for a period of 20 days, starting on PND 75( Day 55 of dosing).
Vaginal smears were also taken on the day of necropsy.

The vaginal smear data was examined to determine the following:
– anomalies of the oestrous cycle;
– the pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Vaginal smears were also performed on the day of necropsy. No vaginal smears were taken from females sacrificed for humane reasons.
Sperm parameters (parental animals):
Seminology and testicular head count - Parental generation, Cohort 1A and Cohort 1B males - All groups
During the necropsy procedure, examination of spermparameters was performed in all males from one epididymal cauda for all P and Cohort 1A males. Spermfrom the cauda was examined for evaluation of motility , morphology and concentration. Spermmotility was evaluated immediately after sacrifice. The percentage of progressively motile spermwas determined. At least 200 spermatozoa per sample were observed and classified as either normal (both head and mid-piece/tail appear normal) or abnormal.

Testicular head count
During the necropsy procedure one testis was taken from all P and Cohort 1A males for enumeration of testicular sperm heads. The testis was decapsulated, weighed and homogenised.
After homogenisation an aliquot of the cell suspension was loaded onto an haemocytometer and resistant spermheads was counted under light microscope.
Spermanalysis and testicular head count of Cohort 1B males were not carried out since no differences were seen between control and treated groups in Parental or Cohort 1A males.
Litter observations:
General litter observations and litter weight
Each litter of the Parental generation was examined, as soon as possible after parturition was considered complete (Day 0 post partum or PND Day 0) to establish the number and sex of pups (live and dead), still births, live births, and the presence of gross anomalies (i.e. externally visible abnormalities, abnormal skin colour or texture; lack of milk in stomach; presence of dried secretions) and a qualitative assessment of body temperature (presence of cold pups), state of activity and reaction to handling. Testis descendent (testes palpable in the scrotum) were checked in all males on Day 21 post partum.
All live pups were individually weighed on Days 1, 4, 7, 14 and 21 post partum. Observations were performed once daily for all litters. Pups found dead were examined at necropsy (external and internal examination) for possible defects and cause of death. All pups sacrificed for humane reasons (external and
internal examinations) were necropsied with the exception of those excessively cannibalised or autolysed.

Adjustment of litter size - Culling - Day 4 post partum
On Day 4 post partum, the size of each litter was adjusted (if possible) by eliminating extra pups by random selection to yield, as nearly as possible, 5 males and 5 females per litter. Selective elimination of pups, e.g. based upon body weight, was not performed.
Whenever the number of male or female pups prevents having five of each sex per litter, partial adjustment (for example, six males and four females) was applied. Litters were not culled to less than 10 pups.

Anogenital distance and presence of nipples/areolae
Anogenital distance
The anogenital distance (AGD) of each pup was measured on Day 1 post partum. The measure of AGD was normalized to the cube root of the pup’s body weight measured on Day 1 post partum.

Nipple count on Day 13 post partum
Male pups
The presence of nipples/areolae was checked on PND 13 and also around PND 21 in case of compound-related findings.

Cohort 1B
General litter observations and litter weight
Each litter of Cohort 1B was examined as soon as possible when parturition was considered complete (Day 0 post partum or PND Day 0) to establish the number and sex of pups (live and dead), stillbirths, live births, and the presence of gross anomalies (i.e. external visible abnormalities, abnormal skin colour or texture; lack of milk in stomach; presence of dried secretions) and a qualitative assessment of body temperature (presence of cold pups), state of activity and reaction to handling.
All live pups were individually weighed on Days 1, 4, 7, 14 and 21 post partum.
Observations were performed once daily for all litters.
Pups found dead or sacrificed for humane reasons were necropsied (external and internal examinations) with the exception of those excessively cannibalised or autolysed.

Adjustment of litter size - Culling on Day 4 post partum
On Day 4 post partum, the size of each litter from Cohort 1B was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, 5 males and 5 females per litter. Selective elimination of pups, e.g. based upon body weight, was not performed.
Whenever the number of male or female pups prevented having five of each sex per litter, partial adjustment (for example, six males and four females) was performed. Litters were not culled to less than 10 pups.

Anogenital distance and presence of nipples/areolae
Anogenital distance
The anogenital distance (AGD) of each pup was measured on Day 1 post partum. The measure of AGD was normalized to the cube root of the pup’s body weight measured on Day 1 post partum.

Nipple count on Day 13 post partum
Male pups
The presence of nipples/areolae was checked on PND 13 and on PND 22 at necropsy.

Testis descendent - F2 males
Testis descendent (testes palpable in the scrotum) was checked in all males at Day 21 post partum.
Postmortem examinations (parental animals):
Terminal studies - (Parental generation,Cohort 1A and Cohort 1B animals)
Euthanasia
P generation, Cohorts 1A and 1B
Adult animals in extremis or killed for humane reasonswere sacrificed under carbon dioxide asphyxiation.
Adult animals that had completed the scheduled test period were killed by exsanguination under isoflurane anaesthesia.
P generation
Males were killed after at least 10 weeks of treatment.
Females with live pups were killed on Day 22 post partum. The females with total litter loss were killed on the day of the occurrence of total litter loss or shortly after. The females showing no evidence of copulation were killed 28 days after the last day of the mating session. The females which do not give birth 25 days after positive identification of mating will be killed shortly after.

Cohort 1A animals were killed at approximately 13 weeks of age.

Cohort 1B
Males were killed after the weaning of all F2 litters. Females with live pups were killed on Day 22 post partum or shortly after.

Pups not selected for blood collection at PND 4
Culled pups not selected for blood collection, pups in extremis or killed for humane reasons and unselected pups were euthanised by intraperitoneal injection of Sodium Thiopenthal.

Pups at PND 4 selected for blood collection
Pups selected for blood collection (F1 or F2) for hormone determination were sacrificed by exsanguination under ether anaesthesia followed by intraperitoneal injection of Sodium Thiopenthal.. Blood was collected by intracardiac puncture.

Pups at PND 22 selected for blood collection
Pups selected for blood collection were euthanised by isoflurane anaesthesia. Blood was collected from vena cava.

Pups at PND 22 not selected for blood collection
Pups not selected for blood collection were killed by carbon dioxide asphyxiation.

Vaginal smears - P generation and Cohorts 1A and 1B females
For adult P and F1 females, a vaginal smear was taken on the day of necropsy and examined to determine the stage of the oestrous cycle. The vaginal smear was not taken in females sacrificed for humane reasons.

Nipple count and retention on Day 22 post partum (F1 and F2 generation)
No nipples were examined since no nipples were observed on PND 13.

Blood samples at necropsy - P generation, pups and Cohort 1A and 1B
P generation
During the necropsy procedure blood samples from the vena cava was taken from 10 randomly selected P males and P females per dose group for coagulation test.
Pups at PND 4
During the necropsy procedure of surplus F1 pups at PND 4 (after culling), blood samples were collected by intracardiac heart puncture for determination of T4 concentrations.
Pups at PND 22
Blood was collected from F1 pups not selected for Cohorts) and F2 on PND 22.
Cohorts 1A
During the necropsy procedure blood samples from the vena cava were taken from selected males and females per dose group for coagulation investigations.

Bone marrow collection - Parental generation, Cohort 1A and Cohort 1B
During the necropsy procedure, shortly after the death of each animal (except for those found dead), bone marrow samples were obtained from the femur of all animals (Parental generation and Cohorts 1A and 1B). Smears prepared from these samples were air dried, fixed in methanol, stained using a May-Grunwald-Giemsa procedure and stored.
In the first instance, bone marrow was evaluated from 10 adult male and 10 adult female animals per group, randomly selected of Cohort 1A. The smears were examined for abnormalities and a differential count was performed including calculation of the myeloid/erythroid cell ratio.
The examination of bone marrow of the parental generation and Cohort 1B animals was not performed since no treatment-related differences were seen in Cohort 1A animals.

Lymph nodes weight and splenic lymphocyte subpopulation – Cohort 1A
From 10 males and 10 females of Cohort 1A of each treatment group (1 male or 1 female per litter; all litters represented by at least 1 pup; randomly selected), axillary and mesenteric lymph nodes were weighed. In addition, the spleen of the same animals was removed and weighed. A portion of spleen was weighed and transferred into appropriate culture medium.
Splenocytes were mechanically separated and the main cell populations T (CD3+), T-helper (CD3+ CD4+), T-cytotoxic (CD3+ CD8+), B (CD45RA+) and NK (CD161+) were identified by means of specific antibodies and flow cytometric acquisition and analysis. The other part of the spleen was preserved in appropriate fixative for histopathological evaluation.

Organ weights - Parental generation and Cohorts 1A and 1B animals
From all animals completing the scheduled test period, the organs indicated in the attached tables were dissected free of fat and weighed. The ratios of organ weight to body weight was calculated for each animal.

Organ weights and tissue preservation - F1 and F2 pups at Day 22 post partum
The pups not selected for cohorts, were sacrificed after weaning (on PND 22). All pups were subjected to gross necropsy (external and internal examination). For at least 10 pups per sex per group, from different litters, if possible, the following organs were weighed and retained in the 10% buffered formalin.
– Brain
– Liver
– Spleen
– Thymus
– Thyroid
Mammary tissues from the same male and female pups were preserved. The hairloss noted at necropsy in pups at PND 22 from dam no. 77 (Group 2) and no. 163 (Group 3) were retained.

Tissues fixed and preserved - Parental generation and Cohorts 1A and 1B animals
Samples of all the tissues listed in the attached tables were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).

Histopathological examination - Parental generation and Cohorts 1A and 1B animals
The tissues required for histopathological examination are listed in the attached tables. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin.

Cohort 1A animals
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
Parental generation and Cohort 1A animals:
The examination was restricted as detailed further below:
– Tissues specified further below from all animals in the control and high dose groups
– Reproductive organs of animals suspected of reduced fertility (e.g. that failed to mate or conceive)
– All abnormalities in all groups

Cohort 1B animals
Histopathological examination was conducted in all males that failed to induce pregnancy and all females non pregnant. The examination was performed on reproductive organs only. The staging of the spermatogenic cycle was also examined.

Enumeration of ovarian follicles - F1 females (Cohort 1A) Histopathological examination including a quantitative evaluation of primordial and small growing follicles, as well as corpora lutea (in one ovary) in Cohort 1A females was performed (HE stained step sections of ovaries and corpora lutea (cut at sections 5 microns thick, and every 20th section (or the section from every 100 micron interval) retained from each half of the ovary, for a total of 5 double sections (or 10 single sections in total)).
The examination was as detailed below:
– From all animals in the control and high dose groups

Presentation of data
Group mean values were calculated for all parameters. Data from non-pregnant females or females with total litter loss, females which did not mate, females with total resorption or from dams without live young were excluded from group mean calculations as considered appropriate by the Study Director or where applicable. The exclusions will be indicated in the relevant Appendix. All derived values (e.g., means, percentages, ratios) were calculated within the litter and the group values expressed as a mean of individual litter values.





Postmortem examinations (offspring):
Terminal studies - (Parental generation,Cohort 1A and Cohort 1B animals)
Euthanasia
P generation, Cohorts 1A and 1B
Adult animals in extremis or killed for humane reasonswere sacrificed under carbon dioxide asphyxiation.
Adult animals that had completed the scheduled test period were killed by exsanguination under isoflurane anaesthesia.
P generation
Males were killed after at least 10 weeks of treatment.
Females with live pups were killed on Day 22 post partum. The females with total litter loss were killed on the day of the occurrence of total litter loss or shortly after. The females showing no evidence of copulation were killed 28 days after the last day of the mating session. The females which do not give birth 25 days after positive identification of mating will be killed shortly after.

Cohort 1A animals were killed at approximately 13 weeks of age.

Cohort 1B
Males were killed after the weaning of all F2 litters. Females with live pups were killed on Day 22 post partum or shortly after.

Pups not selected for blood collection at PND 4
Culled pups not selected for blood collection, pups in extremis or killed for humane reasons and unselected pups were euthanised by intraperitoneal injection of Sodium Thiopenthal.

Pups at PND 4 selected for blood collection
Pups selected for blood collection (F1 or F2) for hormone determination were sacrificed by exsanguination under ether anaesthesia followed by intraperitoneal injection of Sodium Thiopenthal.. Blood was collected by intracardiac puncture.

Pups at PND 22 selected for blood collection
Pups selected for blood collection were euthanised by isoflurane anaesthesia. Blood was collected from vena cava.

Pups at PND 22 not selected for blood collection
Pups not selected for blood collection were killed by carbon dioxide asphyxiation.

Vaginal smears - P generation and Cohorts 1A and 1B females
For adult P and F1 females, a vaginal smear was taken on the day of necropsy and examined to determine the stage of the oestrous cycle. The vaginal smear was not taken in females sacrificed for humane reasons.

Nipple count and retention on Day 22 post partum (F1 and F2 generation)
No nipples were examined since no nipples were observed on PND 13.

Blood samples at necropsy - P generation, pups and Cohort 1A and 1B
P generation
During the necropsy procedure blood samples from the vena cava was taken from 10 randomly selected P males and P females per dose group for coagulation test.
Pups at PND 4
During the necropsy procedure of surplus F1 pups at PND 4 (after culling), blood samples were collected by intracardiac heart puncture for determination of T4 concentrations.
Pups at PND 22
Blood was collected from F1 pups not selected for Cohorts) and F2 on PND 22.
Cohorts 1A
During the necropsy procedure blood samples from the vena cava were taken from selected males and females per dose group for coagulation investigations.

Bone marrow collection - Parental generation, Cohort 1A and Cohort 1B
During the necropsy procedure, shortly after the death of each animal (except for those found dead), bone marrow samples were obtained from the femur of all animals (Parental generation and Cohorts 1A and 1B). Smears prepared from these samples were air dried, fixed in methanol, stained using a May-Grunwald-Giemsa procedure and stored.
In the first instance, bone marrow was evaluated from 10 adult male and 10 adult female animals per group, randomly selected of Cohort 1A. The smears were examined for abnormalities and a differential count was performed including calculation of the myeloid/erythroid cell ratio.
The examination of bone marrow of the parental generation and Cohort 1B animals was not performed since no treatment-related differences were seen in Cohort 1A animals.

Lymph nodes weight and splenic lymphocyte subpopulation – Cohort 1A
From 10 males and 10 females of Cohort 1A of each treatment group (1 male or 1 female per litter; all litters represented by at least 1 pup; randomly selected), axillary and mesenteric lymph nodes were weighed. In addition, the spleen of the same animals was removed and weighed. A portion of spleen was weighed and transferred into appropriate culture medium.
Splenocytes were mechanically separated and the main cell populations T (CD3+), T-helper (CD3+ CD4+), T-cytotoxic (CD3+ CD8+), B (CD45RA+) and NK (CD161+) were identified by means of specific antibodies and flow cytometric acquisition and analysis. The other part of the spleen was preserved in appropriate fixative for histopathological evaluation.

Organ weights - Parental generation and Cohorts 1A and 1B animals
From all animals completing the scheduled test period, the organs indicated in the attached tables were dissected free of fat and weighed. The ratios of organ weight to body weight was calculated for each animal.

Organ weights and tissue preservation - F1 and F2 pups at Day 22 post partum
The pups not selected for cohorts, were sacrificed after weaning (on PND 22). All pups were subjected to gross necropsy (external and internal examination). For at least 10 pups per sex per group, from different litters, if possible, the following organs were weighed and retained in the 10% buffered formalin.
– Brain
– Liver
– Spleen
– Thymus
– Thyroid
Mammary tissues from the same male and female pups were preserved. The hairloss noted at necropsy in pups at PND 22 from dam no. 77 (Group 2) and no. 163 (Group 3) were retained.

Tissues fixed and preserved - Parental generation and Cohorts 1A and 1B animals
Samples of all the tissues listed in the attached tables were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).

Histopathological examination - Parental generation and Cohorts 1A and 1B animals
The tissues required for histopathological examination are listed in the attached tables. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin.

Cohort 1A animals
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
Parental generation and Cohort 1A animals:
The examination was restricted as detailed further below:
– Tissues specified further below from all animals in the control and high dose groups
– Reproductive organs of animals suspected of reduced fertility (e.g. that failed to mate or conceive)
– All abnormalities in all groups

Cohort 1B animals
Histopathological examination was conducted in all males that failed to induce pregnancy and all females non pregnant. The examination was performed on reproductive organs only. The staging of the spermatogenic cycle was also examined.

Enumeration of ovarian follicles - F1 females (Cohort 1A) Histopathological examination including a quantitative evaluation of primordial and small growing follicles, as well as corpora lutea (in one ovary) in Cohort 1A females was performed (HE stained step sections of ovaries and corpora lutea (cut at sections 5 microns thick, and every 20th section (or the section from every 100 micron interval) retained from each half of the ovary, for a total of 5 double sections (or 10 single sections in total)).
The examination was as detailed below:
– From all animals in the control and high dose groups

Presentation of data
Group mean values were calculated for all parameters. Data from non-pregnant females or females with total litter loss, females which did not mate, females with total resorption or from dams without live young were excluded from group mean calculations as considered appropriate by the Study Director or where applicable. The exclusions will be indicated in the relevant Appendix. All derived values (e.g., means, percentages, ratios) were calculated within the litter and the group values expressed as a mean of individual litter values.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n>5.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
Intergroup differences between the control and treated groups was assessed by the non parametric version of theWilliams test. Details of all tests used and the data to which they were applied are included in the study report.
Intergroup differences between the control and treated groups were assessed by the nonparametric version of theWilliams test or with T test. The criterion for statistical significance was p<0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Reproductive indices:
The following reproductive indices were calculated:
Males and Females: Copulatory Index, Fertility Index, Pre−Coital Interval
Offspring viability indices:
Females: Pre-natal loss, Post-natal loss at birth, Post-natal loss at Day 4 post partum (before culling), Post-natal loss through Days 7, 14 and 21 post partum (after culling)
Sex ratios were calculated at birth, Days 4 and 21 post partum and are presented as the percentage of males per litter.
Lactation index (%) for F0 and F1 parental generation
Values for each stage of pup development are presented in terms of days post partum.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
An increased incidence of presence of salivation was noted in all treated males, compared to controls. The incidence was dose-related.
Salivation was noted in one high dose female during post coitum period.
Presence of scabs and/or hairloss observed both in treated and control animals was considered unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Five cases (females) of unscheduled death occurred. Two (animal 131 and 157) at 300 mg/kg/day (humane kill and found dead) and three (animal 177, 191 and 213) at 1000 mg/kg/day (humane kill, died at bleed, found dead).
Macroscopic and microscopic observations consisted of:
– Female no. 131- No macroscopic or microscopic observations were described. The cause of death remained undetermined.
– Female no. 157 - At necropsy dark red fluid with oleous appearance in the thoracic cavity was described. Microscopically alveolar haemorrhage in the lungs was observed. Death was attributed to mis-dosing
– Female no. 177 - At necropsy no findings were noted. Microscopically moderate hepatocytic necrosis in the liver. The cause of death could not be clarified and remained undetermined.
– Female no. 191 - At necropsy observations of thickened mucosa of the stomach, reduced size of the thymus and dark/red colour of the uterus were described. These necropsy observations did not have microscopic correlate. Death was considered related to the bleeding procedure.
– Female no. 213 - At necropsy abnormal size of the spleen (thin). There were no pathology observations for this animal. The cause of death remained undetermined.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight gain of treated animals were compared to controls.
The slight statistically significant lower body weight gain noted on Day 22 in high dose males was considered incidental.
At Day 21 post partum the small reduction in body weight and weight gain of animals including controls was noted and was considered related to the fasting procedure applied for haematological investigations.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment-related changes were seen in food consumption.
The slight statistically significant reduction in food consumption noted in lowdose males on Day 78 of study was considered related to the fasting procedure applied for haematological investigations.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No relevant changes were observed between the control and the treatment groups.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were recorded.

Some statistically significant differences were recorded between control and females dosed at 100 and/or 1000 mg/kg/day: alanine aminotransferase, gamma glutamyl transferase, urea, globulin and potassium. Few animals showed gamma glutamyl transferase values outside the range of historical data; however, since changes were not consistent between sexes, due to individual values and no other related findings were recorded (e.g. hepatic/cholestatic dysfunction), they were considered to be incidental. The other changes were within the range of within the range of expected biological variation and historical data,
therefore they were considered to be incidental.
Endocrine findings:
no effects observed
Description (incidence and severity):
Parental generation
No treatment-related changes were recorded.
TSH was statistically significantly higher than controls in females dosed at 300 mg/kg/day. One animal (no. 129) showed TSH value higher than the limit of historical control data. However, since the observed change was not dose-related, it was considered to be incidental.

F1 pups - Day 4
No treatment-related changes were recorded. T4 was statistically significantly lower than controls in male pups dosed at 1000 mg/kg/day (11%). All animals, with the exception of male pup no. 5 from Dam 173 had T4 value lower than the historical data range. However, values below the range of historical data were also recorded in two control male pups: pup male 7 from dam 17 and pup male 8 from dam 43). In addition changes were minimal and no other related findings were recorded (e.g. thyroid weight changes). Additionally, no compound-related effects were observed in the offspring at Day 22 and in parental animals.
Therefore the differences in T4 were considered to be of no biological relevance. T4 was statistically significantly higher than controls in female pups dosed at 300mg/kg/day. Values were all within the range of historical data and change was not dose-related, therefore it was considered to be incidental.
F1 pups - Day 22
No changes were recorded
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Behavioural observation from removal from the cage and observation in an open field did not show any differences between the control and the treated animals.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changeswere noted in males and females, when compared to controls.
Any microscopic changes were within the range of expected spontaneous findings inWistar Hannover rats of the same age considered incidental and unrelated to the test item.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changeswere noted in males and females, when compared to controls.
Any microscopic changes were within the range of expected spontaneous findings inWistar Hannover rats of the same age considered incidental and unrelated to the test item.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The oestrous cyclicity of the treated females monitored during the pre-mating period, for a total of 14 days, was not affected by treatment. The mean number of oestrous cycles observed in treated animals and controls was comparable.
An oestrous phase was never observed in the female 171 from group 4. This isolated case is considered incidental. The female mated and littered.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Spermmotility, morphology, concentration (expressed as million sperms/gr cauda) and the daily sperm production (expressed ad million sperms/gr testis) were unaffected by treatment.
Reproductive performance:
no effects observed
Description (incidence and severity):
The copulatory index (%) was: 96 in the control group, 93 in the low dose group, 93 in the mid-dose group and 100 in the high dose group.
The fertility index (%) was: 81 in the control group, 92 in the low dose group, 96 in the mid-dose group and 85 in the high dose group.
The number of implantations, live litter size and pre-natal loss did not show treatmentrelated differences. Gestation length periods were similar between groups. All dams gave birth between Days 22 and 24 post coitum.

Details on results (P0)

Fate of females and pregnancy data
Six females in the control group, four females in the low dose groups, three females in the mid-dose group and 4 females in the high dose group were not pregnant.
One mid-dose female (no. 127) lost its litter on Day 1 post partum.
For the low dose female no. 77 the identification of successful mating was not detected.
The animal was separated from the male at the end of the 14 days of mating period and it was found with viable litter a few days later.
Unilateral total resorption was described for low dose female no. 91 sacrificed after 25 days post coitum.
The number of females with live pups on post partum Day 22 were: 22 in the control group, 23 in the low and mid-dose groups and 22 in the high dose group.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs - Cohort 1B
Presence of salivation was noted in almost all high dose males.
Presence of salivation was noted in 5 females during the pre-mating period and one female during the post coitum period. During the post partum period salivation in females was no longer evident.
Presence of scabs and abrasions were noted in the mid-dose male no. 526. Signs started to be evident during last week of treatment. This isolated case is considered incidental.
Tooth/teeth broken/missing was observed in the low dose male no. 478. The presence is considered related to the gavage procedure and not related to treatment.
The low dose female no. 483 on Day 65 of treatment showed presence of staining on muzzle, piloerection, dyspnoea and pallor and was sacrificed for humane reasons. The macroscopic observations revealed that the animal was mis-dosed.
Female no. 515 from the mid-dose group showed marked and diffuse presence of scabs and hairloss during the lactation period and was sacrificed for humane reasons.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Cohort 1B
Two cases (females) of unscheduled death occurred during the study.
Macroscopic and microscopic observations consisted of:
Female 483 - At necropsy of brown staining of muzzle, single abnormal open area in the oesophagus, single, firm, raised with c/s green granular in the right caudal pulmonary lobe and reduced size of the thymus were observed. Death was attributed to misdosing.
Female 515 - At necropsy observation of multiple, scab(s) in the skin of neck, dorsum, head and ventral region, abnormal colour of the thymus and adrenals were described. The illness for this female was related to the integumentary lesion which cause remained
undetermined.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Bodyweight and bodyweight gain - Cohort 1B
On Day 21 of age Day 1 of the dosing phase, before the start of treatment, the body weight of the selected pups was comparable between groups with the exception of the low dose male pups weight which was slight statistically significantly lower, when compared to controls (48.7g versus 51.9g).
A minimal reduction, less than 10% and without a dose relation, was noted in body weight of all treated males compared to controls during the entire treatment period. A slight statistically reduction in body weight was observed in low dose males during the entire
treatment period, when compared to controls.
The body weight of low dose males was statistically significantly lower from Day 1 of treatment to Day 106 (with exception of Day 8), when compared to controls.
The body weight of the high dose males was statistically significantly lower from Day 15 to the end of the treatment period.
Body weight gain of treated males showed a similar lower tendency as the body body weight.
Body weight and body weight gain of treated females was comparable to controls.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption - Cohort 1B
A slight significant reduction in food consumption was observed sporadically in mid-dose males and from Day 29 to the end of the treatment period for the high dose males, when compared to controls.
Food consumption of treated females was similar to controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
see Cohort 1A
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
see Cohort 1A
Endocrine findings:
no effects observed
Description (incidence and severity):
see Cohort 1A
Urinalysis findings:
no effects observed
Description (incidence and severity):
see Cohort 1A
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Detailed clinical observations (FunctionalObservationBatteryTests) -Cohort 1B
Behavioural observation from removal from the cage and observation in an open field did not show any differences between the control and the treated animals.
Immunological findings:
no effects observed
Description (incidence and severity):
see Cohort 1A
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
see Cohort 1A
Gross pathological findings:
no effects observed
Description (incidence and severity):
see Cohort 1A
Neuropathological findings:
not examined
Description (incidence and severity):
see Cohort 1A
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
see Cohort 1A
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
see Cohort 1A

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
see Cohort 1A
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
see Cohort 1A
Reproductive performance:
no effects observed
Description (incidence and severity):
Cohort 1B
The copulatory index (%) was: 100 in the control group, 88 in the low dose group, 84 in the mid-dose group and 92 in the high dose group.
The fertility index (%) was: 96 in the control group, 75 in the low dose group, 84 in the mid-dose group and 96 in the high dose group. Since the fertility index and the copulatory index in the high dose group was similar to controls in the absence of a dose-relation trend
the effects noted in low and mid dose group were not considered treatment-related.

Implantations, pre-natal loss data andg estation length -Cohort 1B
No differences were seen in number of implantations, pre-natal loss and gestation length between the control and the treated females.

Details on results (P1)

Fate of females (Cohort 1B)
One female in the control group (no. 433), 3 females (nos. 451, 461, 473) in the low dose group and 1 female (no. 561) in the high dose group with positive signs of copulation were found not pregnant at necropsy.
Three females in the low dose group (nos. 437, 447, 455), three females (nos. 505, 519, 529) in the mid-dose group and two females (nos. 549, 551) in the high dose group did not mate.
Two dams in the control group (nos. 397, 401), one dam in the low dose group (no. 475), one dam in the mid-dose group (no. 503) and one dam in the high dose group (555) lost it litters on the day of parturition on Day 0/1 post partum.
One control female (no. 427), two mid-dose females (nos. 485, 497) and four high dose females (nos. 543, 553, 563, 583) had unilateral implantation.
The number of females with live pups on post partum Day 21 were: 22 in the control group, 17 in the low dose group, 19 in the mid-dose group and 20 in the high dose group.

Effect levels (P1)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P1)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Clinical signs were described in control and treated pups with similar incidence or without dose relationship and were considered incidental.

Cohort 1A
Salivation was the treatment-related sign observed in almost all males and in 8 out of 20 females of the high dose group.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No differences in total and live litter size, pup loss, litter weights and mean pup weight were observed among treated and control females from birth to PND 22.

Cohort 1A
Four cases (1 male and 3 females) of unscheduled deaths occurred during the study
Macroscopic and microscopic observations consisted of:
– Female no. 253 - Necropsy observation consisted of dark/red colour in the lungs and trachea with clear fluid in the thoracic cavity. Microscopically, moderate haemorrhage was noted in the lungs. Death was attributed to mis-dosing
– Female no. 255 - Necropsy observation of oedematous consistency of the the thymus, single open area in the oesophagus, abnormal clear fluid and white contents (test item like) in the thoracic cavity. These observations corresponded microscopically with lymphoid depletion in the thymus and spleen, acute inflammation with haemorrhage in the surrounding connective tissues of the oesophagus. Death was attributed to mis-dosing
– Female no. 299 - No necropsy observation. The clinical condition, the severe reduced terminal body weight (17.2 gr) and the marked to severe atrophy in the haempoietic tissues (thymus and bone marrow) could be considered unrelated to treatment.
– Male no. 372 - Necropsy observation consisted of abnormal dark red fluid contents in the thoracic cavity. Death was attributed to mis-dosing.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No differences in total and live litter size, pup loss, litter weights and mean pup weight were observed among treated and control females from birth to PND 22.

Cohort 1A
On Day 21 of age Day 1 of the dosing phase, before the start of treatment, the body weight of the selected pups was comparable to controls.
A minimal reduction, less than 10% and without a dose relation, was noted in bodyweight of all treated males compared to controls during the entire treatment period. The reductions were significant from Day 22 of treatment for low dose males and from Day 8 of treatment for high dose males.
Body weight gain of treated males showed a similar lower tendency as the body body weight. The lower body weight gain was significant at statistical analysis on Day 36 for the mid-dose group and on Days 8, 22, 29 and 36 for the high dose group, when compared to controls.
Body weight and body weight gain of of treated females was comparable to controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Cohort 1A
The food consumption of treated animals was slightly reduced compared to controls during the treatment period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Haematology - Cohort 1A
No treatment related changes were recorded. Some statistically significant differences recorded between control and animals dosed at 100 and/or 300 mg/kg/day were recorded, such as: mean corpuscular haemoglobin in males, lymphocytes, eosinophils, basophils and large unstained cells in females. Even though some animals showed values slightly outside of the range of historical data, changes were not dose related, therefore they were considered to be incidental.

Coagulation - Cohort 1A
No changes were recorded.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Clinical chemistry - Cohort 1A
No treatment-related changes were recorded. Some statistically significant differences were recorded between control and treated animals: gamma glutamyl transferase, triglycerides and potassium in males, glucose, protein, albumin and calcium in females). Few values of glutamyl transferase, protein and calcium were outside the range of historical data.
However, changes were not dose-related, not consistent between sexes and/or within the range of the control group data, therefore they were considered to be incidental.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis - Cohort 1A
No changes were recorded.
Sexual maturation:
no effects observed
Description (incidence and severity):
Vaginal opening and first oestrous - Cohort 1A females
The occurrence of vaginal opening was similar between the controls and the treated animals.
The first oestrous was observed on the same day of vaginal opening or within the range of 1-3 days with the exception of one mid-dose female no. 331 from group 7 and female no. 353 from group 8 in which the first occurrence of the oestrous was observed 5 days after the vaginal opening. These two isolated cases were considered incidental.

Balano-preputial skin folds separation - Cohort 1A males
The occurrence of balano preputial separation was similar between the controls and the treated animals.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance was comparable between groups.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No nipples were found in male pups.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Absolute organ weight of pups at PND 22 was comparable between the control and the treated groups.

Terminal body weight and organ weights - Cohort 1A
No treatment-related changes were noted in terminal body weight or absolute or relative organ weights in males and females, when compared to controls. Any terminal body weight or organ weight variations were within the range of expected variations in Wistar Hannover rats of the same age and considered incidental and unrelated to treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Decedent pups
No abnormalities were recorded with the exception of hydrocephaly observed in low dose (pup 18 from dam 79) and an abnormal colour of brain noted in one high dose pup (pup 2 formdam 223) and abnormal colour of brain noted in one high dose pup (pup no. 2 from dam 223).These isolated instances are considered incidental.
Culled pups at Day 4 post partum
No abnormalities were recorded.
Pups at Day 22 post partum
No abnormalities were found with the exception of hairloss noted in a number of pups from the low dose litter no. 77 and the mid-dose litter no. 163 and dark thymus recorded in one low dose pup (no.15) from litter 87. These occurrences without a dose relathion were considered incidental.

Macroscopic observations -Cohort 1A
Any macroscopic observations were within the range of expected spontaneous findings in Wistar Hannover rats of the same age considered incidental and unrelated to the test item.
Histopathological findings:
no effects observed
Description (incidence and severity):
Decedent pups
No abnormalities were recorded with the exception of hydrocephaly observed in low dose (pup 18 from dam 79) and an abnormal colour of brain noted in one high dose pup (pup 2 formdam 223) and abnormal colour of brain noted in one high dose pup (pup no. 2 from dam 223).These isolated instances are considered incidental.
Culled pups at Day 4 post partum
No abnormalities were recorded.
Pups at Day 22 post partum
No abnormalities were found with the exception of hairloss noted in a number of pups from the low dose litter no. 77 and the mid-dose litter no. 163 and dark thymus recorded in one low dose pup (no.15) from litter 87. These occurrences without a dose relathion were considered incidental.

Microscopic observations, spermatogenic cycle - Cohort 1A
No treatment-related changeswere noted in males and females, when compared to controls.
Any microscopic changes were within the range of expected spontaneous findings in Wistar Hannover rats of the same age considered incidental and unrelated to the test item.
No treatment-related effect on the spermatogenic cycle was noted in males, when compared to controls. Qualitative testis staging did not indicate any morphologic abnormalities in the seminiferous epithelium at different stages of the spermatogenic cycle.
Other effects:
no effects observed
Description (incidence and severity):
Sex ratios, expressed as percentage of males, did not show significant differences between groups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Cohort 1A
Behavioural observation from removal from the cage and observation in an open field did not show any differences between the control and the treated animals.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
Bone marrow evaluation - Cohort 1A
No differences were recorded between control and treated animals.

Splenic lymphocyte subpopulation - Cohort 1A
No sign of alteration in the immune cell distribution was observed in splenocytes of animals treated with the test item at any dose level, when compared to controls.

Details on results (F1)

Oestrous cycle - Cohort 1A females
The oestrous cyclicity of the treated females monitored during the last 20 days of dosing was not affected by the treatment.

Thyroid hormone determination - Cohort 1A
No changes were recorded.

Enumeration of ovarian follicles - Cohort 1A
No treatment-related changeswere noted in the differential follicle and corpora lutea counts in the serial ovary sections in females, when compared to controls.

Sperm analysis and daily sperm production - Cohort 1A
Spermmotility, morphology, concentration (expressed as million sperms/gr cauda) and the daily sperm production (expressed ad million sperms/gr testis) were unaffected by treatment.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1A)
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1B)
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
not examined
Description (incidence and severity):
F2 pups
Clinical signs in pups were comparable between the control and the treated groups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted in terminal body weight organ weights.
The higher relative weights of testes and axillary lymph nodes at 1000 mg/kg/day were considered secondary to the lower terminal body weights.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
Balano-preputial skin folds separation - Cohort 1B
The occurrence of balano preputial separation was similar between the controls and the treated animals.

Vaginal opening and first oerstrous - Cohort 1B females
The occurence of vaginal opening was similar between the control and the treated animals
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance of F2 pups
Anogenital distance in male pups was slightly higher (p<005) than the control pups (2.44 versus 2.22 of the control group). The slight difference was considered a normal variation within the expected range in Wistar Hannover rats.
No differences were seen in female pups anogenital distance.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No nipples were found in male pups.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weight of F2 pups
Organ weights in pups at PND 22 was comparable between the control and the treated groups.

No treatment-related changes were noted in terminal body weight organ weights. The higher relative weights of testes and axillary lymph nodes at 1000 mg/kg/day were considered secondary to the lower terminal body weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy findings in F2 pups
Decedent pups or pups killed for humane reasons
No abnormalities were found in pups sacrificed for humane reasons due to the deaths of the dam no. 515.
Major abnormalities were found in one decedent control male pups of dam no. 407 found dead on Day 0 post partum.

Culled pups at Day 4 post partum
No abnormalities were recorded.

Pups at Day 22 post partum
Bent tail was observed in one low dose pup of dam no. 465.
In the mid-dose group:
Thymus, spleen and liver were found small in size for pup no. 2 of dam no. 485
Absence of tail was observed in pup no. 3 of dam no. 489
Abnormal colour of the thymus was noted in pup no. 4 of dam no. 491.
No abnormalities were found in the pups from the high dose group.
Considering that the abnormalities noted in pups were not dose-related the findings were considered incidental.

Macroscopicandmicroscopic observations - Cohort 1B
No treatment-related changes were noted in males and females. Spermatogenic cycle performed in Cohort1B males which failed to induce pregnancy did not showany treatment-related effects.
Histopathological findings:
no effects observed
Description (incidence and severity):
Necropsy findings in F2 pups
Decedent pups or pups killed for humane reasons
No abnormalities were found in pups sacrificed for humane reasons due to the deaths of the dam no. 515.
Major abnormalities were found in one decedent control male pups of dam no. 407 found dead on Day 0 post partum.

Culled pups at Day 4 post partum
No abnormalities were recorded.

Pups at Day 22 post partum
Bent tail was observed in one low dose pup of dam no. 465.
In the mid-dose group:
Thymus, spleen and liver were found small in size for pup no. 2 of dam no. 485
Absence of tail was observed in pup no. 3 of dam no. 489
Abnormal colour of the thymus was noted in pup no. 4 of dam no. 491.
No abnormalities were found in the pups from the high dose group.
Considering that the abnormalities noted in pups were not dose-related the findings were considered incidental.

Macroscopicandmicroscopic observations - Cohort 1B
No treatment-related changes were noted in males and females. Spermatogenic cycle performed in Cohort1B males which failed to induce pregnancy did not showany treatment-related effects.
Other effects:
no effects observed
Description (incidence and severity):
Litter data - Cohort 1B
Litter size, litter weight, mean pup weight and pup loss were unaffected by treatment.

Sex ratios of F2 pups
No difference in sex ratios were seen.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Details on results (F2)

Thyroid hormone determination in F2 pups
No changes were recorded.

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2 (cohort 1B)
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F2)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Discussion

The pre- and post-natal effects of n-Propyl acetate on development, as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring were investigated. In addition, the study provided and/or confirmed information about the effects of the test item on the integrity and performance of the adult male and female reproductive systems.

Parental generation

Groups of 28 male and 28 femaleWistar Han rats received by oral gavage the test item at 0, 100, 300, 1000 mg/kg/day 2 week prior mating (two weeks), mating (two weeks), gestation (three weeks) and lactation (three weeks). An evaluation of male and female reproductive systems was conducted, and included an evaluation of gonadal function, the oestrous cycle, mating performance, conception, gestation, parturition and lactation, as well as survival, growth, and development of the offspring. In life parameters included clinical and detailed clinical observations, feed consumption, body weights, oestrous cyclicity, reproductive performance, pup survival, pup body weights, and puberty onset. Post mortem evaluations in adults included gross pathology, histopathology, organ weight, spermcount, motility and morphology including daily spermproduction. Urinalysis, coagulation, haematology and clinical chemistry were evaluated adult animals (10/sex/group). Hormone panel (T4 and TSH) were evaluated adult animals (10/sex/group) and in 1 pups/sex/litter at Day 4 (T4) and at Day 21 post partum (T4 and TSH). Post mortem evaluations in pups included external and internal examination and organ weights (brain, liver, spleen, thyroid, thymus)

was performed in 10/pups/sex/group on Day 22 post partum.

No mortality related to treatment occurred.

Salivation was the only signs related to treatment observed and mainly in male animals.

No other treatment-related effects were seen in any parameters investigated.

Cohort 1A

Groups of 20 male and 20 female pups were selected from different litters of the parental generation and received by oral gavage the test item at 0, 100, 300, 1000 mg/kg/day from Day 21 of age up to Day 72-78. In life parameters included the onset of vaginal opening, prepurtial separation, clinical and detailed clinical observations, feed consumption, body weights, oestrous cyclicity (performed for at least 2 weeks before and including the day of sacrifice) included gross pathology, histopathology (including staging of the spermatogenic cycle and enumeration of ovarian follicles), organ weight, spermcount, motility and morphology including daily spermproduction. Urinalysis, coagulation, hematology and clinical chemistry were evaluated adult animals (10/sex/group). Hormone panel (T4 and TSH) were evaluated adult animals (10/sex/group).

No mortality related to treatment occurred.

Salivations was the only signs related to treatment observed and mainly in male animals.

A minimal reduction in food consumption, body weight and body weight gain, without dose relation, was observed in male animals compared to controls.

No other treatment-related effects were seen.

Cohort 1B

Groups of 25 male and 25 female pups were selected from different litters of the parental generation and received by oral gavage the test item at 0, 100, 300 and 1000 mg/kg/day 10 prior to breeding (from Day 21 of age), mating (two weeks), gestation (three weeks) and lactation (three weeks). An evaluation of male and female reproductive systems was conducted and included an evaluation of gonadal function, the oestrous cycle, mating performance, conception, gestation, parturition and lactation, as well as survival, growth, and development of the offspring (F2). In life parameters included clinical and detailed clinical observations, feed consumption, body weights, oestrous cyclicity, reproductive performance, pup survival, pup body weights, and puberty onset. Post mortem evaluations in adults included gross pathology and organ weight. Hormone panel (T4 and TSH) were evaluated adult animals (10/sex/group) and in 1 pups/sex/litter at Day 4 (T4) and at Day 21 post partum (T4 and TSH). Post mortem evaluations in pups included external and internal

examination and organ weights (brain, liver, spleen, thyroid, thymus) was performed in 10/pups/sex/group on Day 21 post partum.

No mortality related to treatment occurred.

Salivations was the only signs related to treatment observed and mainly in male animals.

A minimal reduction in food consumption, body weight and body weight gain, without dose relation, was observed in male animals compared to controls.

No other treatment-related effects were seen.

Applicant's summary and conclusion

Conclusions:
Treatment-related effects were noted and and consisted of:
– Salivation mainly observed in males of parental generation, Cohort 1A and Cohort 1B
– Minimal reduction in body weight or body weight gain in males from Cohorts 1A and 1B animals
– Minimal reduction in food consumption in treated animals from Cohorts 1A and 1B animals
The above minimal systemic effects were considered not adverse. No other treatment-related effects were noted.
On the basis of the above results the dosage of 1000 mg/kg/day is considered the NOAEL for systemic toxicity, reproductive toxicity and pup development .