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REACH

Propyl acetate

EC number: 203-686-1 | CAS number: 109-60-4

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

- OECD 443, GLP, 100 - 1000 mg/kg bw/day. NOAEL for systemic toxicity, reproductive toxicity and pups development at 1000 mg/kg/day (2022).

Link to relevant study records

Reference

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
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.
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.
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
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)
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
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.
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: 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.
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
Critical effects observed:: no
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: 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.
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
Critical effects observed:: no
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.
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:: 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.
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
Critical effects observed:: no
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.
Behaviour (functional findings):: not examined
Developmental immunotoxicity:: not examined
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
Critical effects observed:: no
Reproductive effects observed:: no
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 .

Effect on fertility: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 1 000 mg/kg bw/day
Study duration:: subchronic
Species:: rat

Effect on fertility: via inhalation route

Endpoint conclusion:: no study available

Effect on fertility: via dermal route

Endpoint conclusion:: no study available

Additional information

Range-Finder for OECD 443:

The pre- and post-natal effects of n-Propyl on development, as well as the systemic toxicity were investigated in male and female rats of the parental generation orally treated for 4 weeks before pairing and during gestation and lactation periods. Ten male and ten female pups were selected (F1 generation) to evaluate the effect of the test item on offspring, dosed for a total of 7 days from weaning (from Day 21 to Day 28 of age). In addition, reproductive performance such as conception, development of the conceptus and parturition was also examined. Ten male and ten female animals served as controls in parental and F1 generation. Each ten male and ten female animals were dose with n-propyl acetate at 300 and 1000 mg/kg/day in parental and F1 generation.

Parental generation

The following parameters were evaluated: clinical signs, body weight, food consumption, oestrous cycle, mating performance, macroscopic observations and organ weights. In addition, gestation length, sex ratios, pre- and post-natal loss and litter data were evaluated in F0 dams which littered. Litter data and clinical signs of pups from birth to weaning were performed. A post mortem macroscopic observations of pups sacrificed on Day 21 post partum (non selected pups), was also conducted.

F1 generation

Males and females pups weanlings were treated starting from Day 21 of age up to Day 28 and then sacrificed. The following parameters were evaluated: clinical signs, body weight and macroscopic observations.

Parental generation

Mortality and fate of parental females

There was no test item related mortality. The number of females with live pups on Days 21 post partum was 9 in all the groups: control, low dose (300 mg/kg/day) and high dose (1000 mg/kg/day). 1.2Clinical signs

Salivation, considered non-adverse, was occasionally observed for all treated males and in a number of treated females. No clinical signs were observed during the post partum phase.

Body weight and body weight gain

No differences were noted in body weight and body weight gain of treated animals when compared to the control group.

Food consumption

Food consumption was not affected by treatment.

Corpora lutea, number of implantation sites, pre-birth loss data and gestation length of parental females

No treatment-related effects were noted on corpora lutea, number of implantation sites, total litter size at birth and gestation length data of treated females, when compared to the control group.

Litter data

Litter data at birth and throughout the post partum phase up to Day 21 were similar between the control and the treated groups.

Sex ratios of F1 pups

The range of sex ratio did not show differences between groups.

Clinical signs of F1 pups

No compound-related clinical signs were observed

Necropsy findings in F1 decedent pups, humane killed, culled pups on Day 4 post partum and pups sacrificed at weaning

No remarkable findings were detected in decedent pups, and in those sacrificed at weaning (Day 21 post partum).

Terminal body weight and organ weights (F0 generation)

No remarkable changes in terminal body weight and organ weight were observed in treated females when compared to the controls.

Macroscopic observations (F0 generation)

No noteworthy findings were recorded at macroscopic observations.

F1 generation

Mortality and clinical signs No mortality and no clinical signs occurred in F1 animals of both sexes.

Body weight and body weight gain

No differences in mean body weight and body weight gain were observed in treated animals compared to the control group.

Macroscopic observations of F1 animals

No findings were detected at macroscopic evaluation.

Conclusion

Based on the above results, it can be concluded that n-Propyl acetate, orally administered up to the dose level of 1000 mg/kg/day, did not affect gonadal function, mating behavior, conception development of the conceptus and parturition of the parental generation, as well as did not induce effects in the selected offspring (F1 generation), dosed for 7 days from weaning.

OECD 443 (ERBC, 2022)

Summary

Study design

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. The study was conducted according to OECD 443 guideline and GLP (ERBC, 2022). The following parameters were considered: gonadal function, oestrous cycle, epididymal spermmaturation, mating behaviour conception, pregnancy, parturition and lactation.

Males were treated over a 14-day pre-pairing period and during the pairing period and up to one day before necropsy (for a total of at least 70 days of treatment). Females (28/group) were treated throughout over a 14-day pre-pairing period, pairing and gestation periods up to Day 21 post partum. The following dose levels were applied: Group 1: 0 mg/kg body weight/day (control group)

Group 2: 100 mg/kg body weight/day Group 3: 300 mg/kg body weight/day Group 4: 1000 mg/kg body weight/day.

A standard dose volume of 4 mL/kg body weight was used. Control animals were dosed with the vehicle alone (Corn oil).

On post-natal (PND) Day 21, 45 F1 pups/sex/group were randomly selected to serve as Cohort 1A (20/sex/group) and Cohort 1B (25/sex/group).

The treatment of selected pups started on the day of selection, PND 21. The dose levels and the vehicle used for both Cohorts were the same as for the Parental generation.

Cohort 1A animals were treated up to the day before necropsy for at least 72 days. Cohort 1B animals were treated over 72-77 days and then mated. Treatment of males

continued during the mating period up to the sacrifice of the majority of litters (at least 130 days). Treatment of females continued during the mating, gestation and post partum

periods.

Parental generation

The following examinations were performed in adult animals:

Daily clinical signs, detailed clinical observations (FOB), body weight, food consumption, oestrous cycle was monitored starting 2 weeks before mating, mating performance, clinical pathology investigations including urine in 10 males/group and in 10 females/group which littered.

Parental females were allowed to litter and rear their offspring, delivery process was monitored.

Each litter was observed daily and on Day 13 of lactation, mammary areas of male pups was observed to check for presence of nipple. On Day 4 of lactation litters were culled to 10 pups. Pups were individually weighed on post-natal (PND) Days 1, 4, 7, 14 and 21).

At necropsy a detailed macroscopic observation was performed in all parental animals.

Evaluation of sperm analysis, daily sperm production and sperm morphology was performed in all males. Microscopic examination was performed in all control and high dose parental animals. At necropsy all pups were examined externally and internally, the sex was confirmed by internal gonads inspection and at PND 22 brain, liver, thymus, thyroid and spleen were weighed in 10 pups/sex selected from different litters. Thyroid hormones were determinated in 10/sex/group of parental animals (T4 and TSH) and in 10 pup/sex/ from different litters on PND 4 (T4) and on PND 22 (T4 and TSH).

Cohort 1A

On PND 21, 20 pups/sex/group were selected and allocated to the Cohort 1A.

The following examinations were performed:

Daily clinical signs and detailed clinical observations (FOB), body weight, food consumption, oestrous cycle (monitored 2 weeks before the necropsy including the day of sacrifice), clinical pathology investigations, urine and thyroid panels (T4 and TSH). At necropsy, a detailed macroscopic observation was performed in all animals and the organs weighed.

One testis and one epididymis were used for the evaluation of spermanalysis, daily sperm production and spermmorphology for all males.

Evaluation of bone marrow(10/animal/sex/group), microscopic examination including the stages of the spermatogenic cycle and the enumeration of ovarian follicles were evaluated (control and high dose animals).

Cohort 1B

On PND 21, 25 pups/sex/group were selected and allocated to Cohort 1B.

The following examinations were performed:

Parental females were allowed to litter and rear their offspring, delivery process was monitored. Each litter was observed daily and on Day 13 of lactation, mammary areas of male pups was observed to check for presence of nipple. On Day 4 of lactation litters were culled to 10 pups. Pups were individually weighed on post-natal (PND) Days 1, 4, 7, 14 and 21).

At necropsy a detailed macroscopic observation was performed in all parental animals.

At necropsy all pups were examined externally and internally, the sex was confirmed by internal gonads inspection and at PND 22 brain, liver, thymus, thyroid and spleen were weighed in 10 pups/sex selected from different litters. Thyroid hormones were determined in 10/sex/group of parental females (T4 and TSH) and 10 pups/sex selected from different litters on PND 4 (T4) and on PND 22 (T4 and TSH). At necropsy a detailed macroscopic observation was performed in all parental animals and the organs weighed. At necropsy pups were examined externally and internally, the sex was confirmed by internal gonads inspection.

Parental generation (F0)

Mortality - Parental generation

Five cases of unscheduled death occurred during the study: 2 females of the mid-dose group (nos. 131 and 157) and 3 high dose females (nos. 177, 191, 213).

The cause of death for females 131, 177 and 213 remained undetermined.

The cause of death for female no. 157 was attributed to a mis-dosing.

The cause of death for female 191 was considered related to the bleeding procedure.

Fate of females - Parental generation

Non pregnant females were present both in the control and the treated groups with similar incidence. The single occurrence of total litter loss in group 3 was considered incidental.

The number of females with live pups at post partum Day 22 were: 22 in the control and high dose groups and 23 in the low and mid-dose groups.

Clinical signs - Parental generation

An increased incidence of salivation was noted in the treated animals, when compared to controls. The incidence was dose-related.

Salivation was observed in one high dose female during post coitum period.

No other treatment-related clinical signs were observed.

Detailed clinical observations - Parental generation (Functional Observation Battery Tests) of Parental generation

Detailed clinical observations performed atweekly intervals did not revealed any treatmentrelated effects.

Body weight and body weight gain - Parental generation

No treatment-related changes were seen.

Food consumption - Parental generation

No treatment-related changes were seen.

Haematology, coagulation, clinical chemistryandurinalysis -Parental generation

No treatment-related changes were seen.

Thyroid hormone determination of Parental generation - Parental generation and F1 pups

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: male pup 7 from dam 17 and male pup 8 from dam 43. In addition, changes were minimal and no other related findings were recorded (e.g. thyroid weight changes). Additionally T4 levels were not affected in parental animals and in pups on Day 22. 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 300 mg/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.

Oestrous cycle, reproductive parameters and mating performance - Parental generation

Oestrous cycle, pre-coital interval, copulatory and fertility indices of treated animals were comparable to controls.

Implantation sites, pre-natal loss dataandgestation length -Parental generation

Implantations, litter size, pre-natal loss and gestation length were unaffected by treatment.

Litter data and pup loss - Parental generation

No treatment-related effects in litter size, pup loss, litter weight an mean pup weight

Sex ratios of F1 pups

Sex ratios was unaffected by treatment.

Clinical signs of F1 pups and nipple observations

No treatment-related clinical signs were noted in treated pups compared to controls.

No Nipples were found in any pups.

Anogenital distance (AGD) of F1 pups

Anogenital distance was unaffected by treatment.

Necropsy findings of F1 pups

No treatment-related findings were described.

Spermanalysis of - Parental males

No differences in sperm motility, sperm concentration or sperm abnormalities were found between the treated and the control animals.

Daily spermproduction was similar between the control and the treated males.

Organ weights of F1 pups on Day 22 post partum

No differences in pups organ weights were observed.

Terminal body weight and organ weights - Parental generation

No treatment-related changes were noted.

Macroscopic observations - Parental generation

No treatment-related changes were noted.

Microscopic observations - Parental generation

No treatment-related changes were noted.

Cohort 1A

Mortality - Cohort 1A

Four cases of unscheduled death occurred during the study: 2 control females (253, 255), one low dose female (299) and and one high dose male (372).

Male 372 - Death was attributed to misdosing

Female 253 - Death was attributed to misdosing

Female 255 - Death was attributed to misdosing

Female 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.

Clinical signs - Cohort 1A

Salivation was the treatment-related sign observed in 90% of high dose males and 40% of the high dose females.

Detailed clinical observations (FunctionalObservationBatteryTests) -Cohort 1A

Behavioural observation performed during the removal from the cage and in observations in an open field were unaffected by treatment.

Body weight and body weight gain - Cohort 1A

On the day of selection, PND 21, before the start of treatment, the body weight of the selected pups was comparable to controls.

A treatment-related minimal reduction without dose relation, was noted in body weight of all treated males compared to controls during the entire treatment period. The reduction was statistically significant for the low and high dose males.

Body weight gain of males showed a similar trend of body weight. The significance was observed in one occasion for mid-dose males and in a few occasions for high dose males, when compared to controls.

Body weight and body weight gain of females were unaffected by treatment.

Food consumption - Cohort 1A

The food consumption was slightly reduced in treated animal, when compared to controls.

Vaginal opening and first oestrous and balano-preputial skin folds separation - Cohort 1A

No differences related to treatment were seen for the onset of vaginal opening or balano skin folds separation.

Oestrous cycle - Cohort 1A

Oestrous cyclicity was unaffected by treatment.

Haematology, clinical chemistry, coagulation and urinalysis - Cohort 1A

Changes noted in haematology were not dose-related and considered incidental.

No changes were noted in a coagulation, clinical chemistry or urinalysis.

Bone marrow evaluation - Cohort 1A

No differences were noted in bone marrow evaluation between the control and the treated animals.

Thyroid hormone determination - Cohort 1A

No treatment-related changes were recorded.

Spermanalysis and daily spermproduction - Cohort 1A

No differences in epididymal sperm motility, morphology or concentration were seen between the control and the treated animals.

Daily spermproduction was unaffected by treatment.

Terminal body weight and organ weights - Cohort 1A

No treatment-related changes were noted.

Macroscopic observations - Cohort 1A

No treatment-related changes were noted.

Microscopic observations - Cohort 1A

No treatment-related changes were noted.

Spermatogenic cycle - Cohort 1A

No morphological abnormalities were seen in the seminiferous epithelium at different stages of the spermatogenic cycle between the control and the high dose males.

Enumeration of ovarian follicles - Cohort 1A

No treatment-related changes were seen.

Lymph nodes weight and splenic lymphocyte subpopulation - Cohort 1A

No treatment-related differences were seen.

Cohort 1B

Mortality and fate of females - Cohort 1B

Two cases of unscheduled death occurred during the study: 1 low dose female (483) and one mid-dose female (515).

Female 483 - Death was attributed to mis-dosing

Female 515 - Female was humanely killed for integumentary lesions observed in several body areas the cause of which remained undetermined.

Clinical signs - Cohort 1B

Salivation was the treatment-related clinical sign observed in almost all high dose males and in a few females during the premating and post coitum periods.

Detailed clinical observations (FunctionalObservationBatteryTests) -Cohort 1B

Behavioural observation performed during the removal from the cage and observations in an open field were unaffected by treatment.

Body weight,body weight gain and food consumption - Cohort 1B

A treatment-related minimal reduction, was noted in body weight of all treated males compared to controls during the entire treatment period. Body weight gain showed a similar trends with a few occasions statistically significant and only in mid- and high dose males, when compared to controls.

Body weight and body weight gain of females were unaffected by treatment.

Vaginal opening and first oestrous and balano-preputial skin folds separation - Cohort 1B

No differences were seen in the occurrence of vaginal opening or balano preputial separation between the control and the treated animals

Oestrous cycle, reproductive parameters, pairing combination and mating performance - Cohort 1B

Oestrous cycle was unaffected by treatment

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.

Implantation sites, pre-natal loss data and gestation length - Cohort 1B

Number of implantation, pre-natal loss and gestation length were unaffected by treatment.

Litter data - Cohort 1B

Litter data of treated dams was comparable to controls.

Sex ratios of F2 pups

Sex ratios was unaffected by treatment.

Clinical signs and nipple observations of F2 pups

No treatment-related clinical signs were seen in pups. No nipples were found in males pups.

Anogenital distance of F2 pups

No changes attributable to treatment were seen.

Necropsy findings in F2

No treatment-related finding were noted.

Organ weight of F2 pups

Organ weights of treated pups were comparable to the controls.

Thyroid hormonedetermination in F2 pups-Day 4 and Day 22 post partum

No changes were recorded.

Terminal body weight and organ weights - Cohort 1B

No treatment-related changes were noted.

Macroscopic and microscopic observations - Cohort 1B

No treatment-related changes were noted. Spermatogenic cycle performed in Cohort1B males which failed to induce pregnancy did not show any treatment-related effects.

Conclusions

Treatment-related effects were noted 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 as not adverse. No other treatmentrelated effects were noted.

On the basis of the above results the dosage of 1000 mg/kg/day is considered to be the NOAEL for systemic toxicity, reproductive toxicity and pups development.

Effects on developmental toxicity

Description of key information

- OECD 414, GLP, 25 female rats/dose, 100 - 1000 mg/kg bw/day, exposure from GD 6 -19. No maternal and fetal developmental toxicity (2018)

- OECD 414, GLP, 24 female rabbits/dose, 100 - 1000 mg/kg/bw/day, exposure from GD 7 -27. No maternal and fetal developmental toxicity (2019)

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep - Oct 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Jan 2001

Deviations:

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 1998

Deviations:

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2008

Deviations:

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht

Limit test:

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: BASF SE, Ludwigshafen, Germany
- Batch No.of test material: 83294456P0
- Expiration date of the batch: 08 Nov 2018
- Purity: 99.9%
- Identitiy: Confirmed
- Physical state/appearance: liquid/colorless, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions (in the vehicle): Guaranteed by analytical verifications in corn oil over a period of seven days at room temperature
- Homogeneity: given (visually)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature. For the test substance preparations, the specific amount of test substance was weighed, topped up with corn oil in a graduated flask and intensely mixed with a magnetic stirrer or by shaking until it was completely dissolved. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI[Han]

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 144.5 - 195.6 g
- Fasting period before study: no
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (6 am to 6 pm illumination)

IN-LIFE DATES: From: 1st Cohort: 28 Sep 2017; 2nd Cohort: 29 Sep 2017 To: 1st Cohort: 18 Oct 2017; 2nd Cohort: 19 Oct 2017

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Volume applied: 4ml/kg

DIET PREPARATION
- Rate of preparation of diet (frequency): at the beginning and at intervals thereafter
- Mixing appropriate amounts: test substance was weighed, topped up with corn oil and intensely mixed.
- Storage temperature: room temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): non given

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance preparations were sent thrice to the analytical laboratory for verification of the concentrations. Control procedures were Gas chromatography (GC) and H-NMR spectrum.

Details on mating procedure:

Animals were paired by the breeder ("time-mated"); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”.

Duration of treatment / exposure:

GD 6-19

Frequency of treatment:

daily

Duration of test:

14 days

Dose / conc.:

100 mg/kg bw/day

Remarks:

2.5 g/100 ml

Dose / conc.:

300 mg/kg bw/day

Remarks:

7.5 g/100 ml

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

25.00 g/100 ml

No. of animals per sex per dose:

25 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Reason for species selection:
The Crl:WI(Han) strain was selected since extensive historical control data is available from the test facility for Wistar rats. This specific strain has been proven to be sensitive to substances with a teratogenic potential.

Maternal examinations:

MORTALITY: Yes
- Time schedule: twice daily (Mo-Fr), once a day (Sat, Sun)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily before and after treatment

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19, and 20

FOOD CONSUMPTION: Yes
- Time schedule: GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

WATER CONSUMPTION: No data
- Time schedule for examinations: no data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Gross pathology

NECROPSY:
- uteri and ovaries were analyzed: weight of the unopened uterus, number of corpora lutea, number and distribution of implantation sites classified as live fetuses and dead implantations

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Dead fetuses: Yes

Fetal examinations:

- External examinations: Yes:
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: No data
- Weight, sex, external tissues, and all orifices were examined
- Viability
- Condition and weight of placentas, umbilical cords, fetal membranes, and fluids

Statistics:

Food consumptiona), body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight: Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) forthe hypothesis of equal means

Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions

Proportions of fetuses with malformations, variations and/or unclassified observations in each litter: Pairwise comparison of each dose group with the control group using the WILCOXONtest (one-sided) for the hypothesis of equal medians

Indices:

Conception rate, preimplantation loss, postimplantation loss

Historical control data:

MATERNAL BODY WEIGHTS (RANGE) FROM 973 (Days 1 - 20) AND 974 (Day 0) ANIMALS

Day 0: 140.0 - 196.6 g
Day 1: 152.1 - 208.6
Day 3: 158.7 - 216.0
Day 6: 167.6 - 231.5
Day 8: 174.3 - 242.1
Day 10: 181.6 - 252.9
Day 13: 186.6 - 278.4
Day 15: 192.5 - 290.4
Day 17: 183.3 - 318.4
Day 19: 190.5 - 349.8
Day 20: 193.2 - 364.3

REPRODUCTION DATA

- Females mated: 999 of which pregnant: 976 (98%)
- Aborted: 0
- Premature births: 0
- Dams with viable fetuses: 973
- Dams with all resorptions: 3
- Female mortality: 0%
- Corpora lutea (range): 10.5 - 12.3
- Implantations sites (range): 9.9 - 11.8
- Preimplantation loss (range): 1.4 - 13.3%
- Postimplantation loss (range): 3.2 - 14.0%
- Dams with resorptions: 449 (46%)
- Resorptions (range): 0.3 - 1.5
- Dead fetuses: 0
- Viable fetuses: 9778
- Litter size (range): 9.3 - 11.2
- Placenta weights (range): 0.35 - 1.14 g (males); 0.32 - 1.10 g (females)
- Fetal weights (range): 2.8 - 4.5 g (males); 2.3 - 4.4 g (females)

FETAL MALFORMATIONS:

- External malformations: 13 (9778 evaluated) ; 0.1%
- External variations: 4 (9778 evaluated); 0.04%
- External unclassified findings: 3 (9778 evaluated); 0.03%
- Visceral malformations: 13 (4650 evaluated); 0.3%
- Visceral variations: 156 (4650 evaluated); 3.4%
- Visceral unclassified findings: 1 (4650 evaluated); 0.02%
- Skeletal malformations: 39 (5128 evaluated); 0.8%
- Skeletal variations: 5035 (5128 evaluated); 98.2%
- Skeletal cartilage: 3655 (5218 evaluated); 71.3%

Clinical signs:

no effects observed

Description (incidence and severity):

In 21 out of 25 females occasionally salivation during treatment period in group 1000 mg/kg bw/day. Salivation occurred in the respective animals only shortly, i.e. within 0-2 hours, after treatment and was observed during GD 6-8 and GD 15-19. No clinical signs or changes of general behavior were detected in any female of all test groups beyond 2 hours after treatment. The occasional salivation was most probably caused by the bad taste or smell of the test substance and was not assessed as a sign of systemic toxicity.

No other effects observed

Mortality:

no mortality observed

Description (incidence):

No test substance-related or spontaneous deaths in any females.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights and average body weight gain of treated groups comparable to control.

The statistically significantly decreased body weight gain value in test group 1 during GD 3-6 (pre-treatment period) was assessed as incidental.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean food consumption of treated groups comparable to control.

The statistically significantly decreased food consumption value in the mid-dose dams during GD 17-19 was not considered biologically relevant due to the lack of dose response relationship.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Mean gravid uterus weights not influenced by the test substance. Difference between treated groups and control group revealed no dose-dependency and were assessed to be without biological relevance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Three spontaneous findings were noted in three individual females of test groups 0 and 3 (0 and 1000 mg/kg bw/d). These gross findings were:
• Dilated renal pelvis (right) in dam No. 2,
• Diaphragmatic hernia in dam No. 4,
• Stomach: erosion(s) (one) in dam No. 79.
No further necropsy findings which could be attributed to the test substance were seen in any dam.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No test substance related differences in conception rates, mean number of corpora lutea and implantation sites.
Two females in the control group and two females in the 300 mg/kg bw/day group did not get pregnant.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no maternal developmental toxicity observed

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Changes in sex ratio:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

Three external malformations were exclusively detected in the control group.

No fetal external variations were reported. No fetal external unclassified findings were recorded.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were noted in two fetuses of the control group and in one fetus, each, of test groups 1 and 3 (100 and 1000 mg/kg bw/d). One control fetus had associated external malformations. The incidences of these malformations were neither
statistically significantly different from control nor dose-dependent. The finding bipartite basisphenoid occurred in each one fetus of test groups 1 and 3 without relation to dose. The mean value of the incidences in test group 3 was within the historical
control range. Therefore, it was not assessed as treatment-related, adverse finding.
All other malformations in test group 1 occurred in only one fetus (33-10 F) without relation to dose and were single, isolated cases which were mostly covered by the historical control data.
The body weight of this fetus (3.2 g) was lower compared to its group mean value (females of test group 1: 3.5 g). The findings in this fetus were not assessed as treatment-related and adverse.

For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dose. The
overall incidences of skeletal variations were comparable to the historical control data.

Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the ribs and the sternum and did not show any relation to dosing.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Soft tissue malformations were recorded for two control, one mid-dose and three high-dose fetuses. The finding situs inversus in the fetuses of test groups 300 mg/kg bw/day and 1000 mg/kg bw/day were single events in individual fetuses and the mean values were within the historical control data. The findings absent subclavian and anophthalmia were observed in one fetus each of test group 1000 mg/kg bw/day and the mean values were within the historical control ranges.
All these findings were single cases, they were within the historical control ranges.
No ontogenetic pattern is recognizable for all these individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring. Therefore, the observed malformations were not assessed as treatment-related, adverse findings.

Three soft tissue variations were detected, i.e. malpositioned subclavian origin, dilated renal pelvis and dilated ureter. These variations were neither significantly different from the concurrent control nor dose-dependently altered. All of them can be found in the historical control data at comparable incidences. Therefore, they were not assessed as treatment-related.

No fetal visceral unclassified findings were reported.

Other effects:

no effects observed

Description (incidence and severity):

Mean placental weights comparable among groups.

Details on embryotoxic / teratogenic effects:

No teratogenic effects due to treatment were recorded.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No developmental toxicity and teratogenic effects

Abnormalities:

no effects observed

Developmental effects observed:

RESULTS

ANALYSES

Stability analysis

The stability of the test substance in corn oil over a period of 7 days at room temperature was

demonstrated before the start of the study.

Homogeneity analysis of the test substance preparations

Given that the test substance is completely miscible with corn oil, solutions were considered to

be homogenous without further analysis.

Concentration control analysis of the test substance preparations

The results of the analysis of the test substance preparations confirmed the correctness of the

prepared concentrations of the mid- and high-dose (samples 04 and 05, respectively). These

analytical values of the samples corresponded to the expected values within the limits of the

analytical method, i.e. were always above 90% and below 110% of the nominal concentrations.

Concerning the lowest dose (100 mg/kg bw/d), the values of samples 03, 03R and 08 new

(149.8, 139 and 124%, respectively) were outside the range of 90% - 110% of the nominal

concentrations. A mean value for all three measurements of around 138% corresponds to actual

dose of 138 mg/kg bw/d for the lowest test group. This has no influence of the validity of

the study.

Food analyses

On the basis of the duration of use and the analytical findings with respect to chemical and

microbiological contaminants, the food was found to be suitable. The EPA Fed. Reg. of 09 May

1979 (Vol. 44, No. 91, p. 27354) served as a guideline for maximum tolerable chemical contaminants.

The amount of microorganisms did not exceed 1*105/g feed.

The individual results are found in the archives of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany.

Drinking water analyses

On the basis of the analytical findings, the drinking water was found to be suitable. The German

Drinking Water Regulation (“Trinkwasserverordnung”) served as the guideline for maximum

tolerable contaminants.

The individual results are found in the archives of Experimental Toxicology and Ecology, BASF

SE, Ludwigshafen, Germany.

Bedding and enrichment analyses

On the basis of the analytical findings, the bedding and the enrichment were found to be suitable.

Levels given in Lab Animal, Nov-Dec 1979, pp. 24-34, served as a guideline for maximum

tolerable contaminants.

The individual results are found in the archives of Experimental Toxicology and Ecology, BASF

SE, Ludwigshafen, Germany.

Clinical examinations of the dams

Only pregnant dams were used for the calculations of mean maternal food consumption, body

weight and body weight change. Only pregnant dams with scheduled sacrifice (GD 20) were

used for the calculation of mean gravid uterine weights, corrected (net) body weight gain and

summary of reproduction data.

The following females were excluded from the above-mentioned calculations:

Test group 0 (0 mg/kg bw/d):

• Females Nos. 13, 14 – not pregnant

Test group 2 (300 mg/kg bw/d):

• Females Nos. 54, 62 – not pregnant

4.2.1.1. Mortality

There were no test substance-related or spontaneous mortalities in any females of all test

groups (0, 100, 300 or 1000 mg/kg bw/d).

Clinical symptoms

Nearly all (21 out of 25) high-dose females (1000 mg/kg bw/d) showed occasionally salivation

during the treatment period. Salivation occurred in the respective animals only shortly, i.e.

within 0-2 hours, after treatment and was observed during GD 6-8 and GD 15-19. No clinical

signs or changes of general behavior were detected in any female of all test groups beyond 2

hours after treatment. The occasional salivation was most probably caused by the bad taste or

smell of the test substance and was not assessed as a sign of systemic toxicity.

No further clinical signs or changes of general behavior, which may be attributed to the test

substance, were detected in any female at dose levels of 100, 300 or 1000 mg/kg bw/d during

the entire study period.

Food consumption

The mean food consumption of the high-, mid- and low-dose dams (1000, 300 and 100 mg/kg

bw/d) was generally comparable to the concurrent control group throughout the entire study

period.

The statistically significantly decreased food consumption value in the mid-dose dams during

GD 17-19 was not considered biologically relevant due to the lack of dose response relationship.

Body weight data

The mean body weights and the average body weight gain of the low-, mid- and high-dose

dams (100, 300 and 1000 mg/kg bw/d) were generally comparable to the concurrent control

group throughout the entire study period.

The statistically significantly decreased body weight gain value in test group 1 during GD 3-6

(pre-treatment period) was assessed as incidental.

Corrected (net) body weight gain

The corrected body weight gain of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d)

revealed no difference of any biological relevance to the corresponding control group.

Moreover, mean carcass weights of all test groups remained unaffected by the treatment.

Terminal examinations of the dams

Uterus weight

The mean gravid uterus weights of the animals of test groups 1-3 (100, 300 and 1000 mg/kg

bw/d) were not influenced by the test substance. The differences between these groups and

the control group revealed no dose-dependency and were assessed to be without biological

relevance.

Necropsy findings

Three spontaneous findings were noted in three individual females of test groups 0 and 3 (0

and 1000 mg/kg bw/d). These gross findings were:

• Dilated renal pelvis (right) in dam No. 2,

• Diaphragmatic hernia in dam No. 4,

• Stomach: erosion(s) (one) in dam No. 79.

No further necropsy findings which could be attributed to the test substance were seen in any

dam.

Reproduction data

The conception rate was 92% in the control and the mid-dose groups (0 and 300 mg/kg bw/d)

and 100% in the low- and high-dose groups (100 and 1000 mg/kg bw/d). With these rates, a

sufficient number of pregnant females were available for the purpose of this study (according

to the test guidelines listed in chapter 2.3.).

There were no test substance-related and/or biologically relevant differences between the different

test groups in conception rate, in the mean number of corpora lutea and implantation

sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions

and viable fetuses. All observed differences are considered to reflect the normal

range of fluctuations for animals of this strain and age.

EXAMINATION OF THE FETUSES

Sex distribution of the fetuses

The sex distribution of the fetuses in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) was

comparable to the control fetuses.

Weight of the placentae

The mean placental weights of test groups 1-3 were comparable to the concurrent control

group.

Weight of the fetuses

The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance

and did not show any biologically relevant differences in comparison to the concurrent control

group.

Fetal external malformations

Three external malformations were exclusively detected in the control group. In one case, these

external malformations were associated with skeletal malformations. No external malformations

occurred in treated animals.

Tab. 2: Individual fetal external malformations

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	9-03 F	umbilical hernia
0 (0 mg/kg bw/d)	15-03 Fa)	gastroschisis, ectrodactyly
1 (100 mg/kg bw/d)	none
2 (300 mg/kg bw/d)	none
3 (1000 mg/kg bw/d)	none

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

a)fetus with additional skeletal malformations

Tab.3: Total external malformations

Test group0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

LitterFetuses

N N

251

248

229

273

Fetal incidence

N (%)

2 (0.8)

0.0

Litter incidence

N (%)

2 (8.7)

0.0

Affectedfetuses/litter

Mean%

0.8

0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Fetal external variations

No external variations were recorded.

Fetal external unclassified observations

No external unclassified observations were recorded.

Fetal soft tissue malformations

Soft tissue malformations were recorded for two control, one mid-dose and three high-dose

fetuses

The finding situs inversus in the fetuses of test groups 2 and 3 were single events in individual

fetuses and the mean values were within the historical control data (affected fetuses per litter, mean: 0.1%, range of 0.0 - 1.4%).

The findings absent subclavian and anophthalmia were observed in one fetus each of test

group 3 and the mean values were within the historical control ranges (anopthalmia: litters,

range of 0.0 - 4.0%, absent subclavian: affected fetuses per litter, range of 0.0 - 1.1%).

All these findings were single cases, they were within the historical control ranges. No ontogenetic

pattern is recognizable for all these individual malformations nor was there any cluster of

any of these individual malformations seen in the other offspring.

Therefore, the observed malformations were not assessed as treatment-related, adverse

findings.

Tab. 4: Individual soft tissue malformations

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	12-02 F	interrupted spinal cord
0 (0 mg/kg bw/d)	25-14 F	hydronephrosis, hydroureter
1 (100 mg/kg bw/d)	none
2 (300 mg/kg bw/d)	72-06 M	situs inversus
3 (1000 mg/kg bw/d)	76-08 F	situs inversus
	86-04 F	absent subclavian
	95-09 M	anophthalmia

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

Tab. 5: Total soft tissue malformations

Test group0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

LitterFetuses

N N

122

117

110

130

Fetal incidence

N (%)

2 (1.6)

0.0

1 (0.9)

3 (2.3)

Litter incidence

N (%)

2 (8.7)

0.0

1 (4.3)

3 (12)

Affectedfetuses/litter

Mean%

1.5

0.0

0.9

2.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Fetal soft tissue variations

Three soft tissue variations were detected, i.e. malpositioned subclavian origin, dilated renal

pelvis and dilated ureter. These variations were neither significantly different from the concurrent

control nor dose-dependently altered. All of them can be found in the historical control data

at comparable incidences. Therefore, they were not assessed as treatment-related.

TAb. 6: Total soft tissue variations

Test group0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

LitterFetuses

N N

122

117

110

130

Fetal incidence

N (%)

3 (2.5)

2 (1.7)

2 (1.8)

4 (3.1)

Litter incidence

N (%)

3 (13)

2 (8.0)

2 (8.7)

4 (16)

Affectedfetuses/litter

Mean%

2.6

1.3

2.2

3.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Fetal soft tissue unclassified observations

No soft tissue unclassified observations were recorded.

Fetal skeletal malformations

Skeletal malformations were noted in two fetuses of the control group and in one fetus, each,

of test groups 1 and 3 (100 and 1000 mg/kg bw/d). One control

fetus had associated external malformations. The incidences of these malformations were neither

statistically significantly different from control nor dose-dependent.

The finding bipartite basisphenoid occurred in each one fetus of test groups 1 and 3 without

relation to dose. The mean value of the incidences in test group 3 was within the historical

control range (HCD: affected fetuses per litter, range of 0.0 – 0.7%). Therefore, it was not

assessed as treatment-related, adverse finding.

All other malformations in test group 1 occurred in only one fetus (33-10 F) without relation to

dose and were single, isolated cases which were mostly covered by the historical control data.

The body weight of this fetus (3.2 g) was lower compared to its group mean value (females of

test group 1: 3.5 g). The findings in this fetus were not assessed as treatment-related and

adverse.

TAb. 7: Individual fetal skeletal malformations

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	11-04 F	malpositioned and bipartite sternebra
0 (0 mg/kg bw/d)	15-03 Fa)	absent forepaw phalanx, severely malformed sternum
1 (100 mg/kg bw/d)	33-10 F	bipartite basisphenoid, misshapen presphenoidal, severely malformed vertebral column and/or ribs
2 (300 mg/kg bw/d)	none
3 (1000 mg/kg bw/d)	91-07 M	bipartite basisphenoid

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

a)fetus with additional external malformations

Tab.8: Total skeletal malformations

Test group0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

LitterFetuses

N N

129

131

119

143

Fetal incidence

N (%)

2 (1.6)

1 (0.8)

0.0

1 (0.7)

Litter incidence

N (%)

2 (8.7)

1 (4.0)

0.0

1 (4.0)

Affectedfetuses/litter

Mean%

2.0

0.8

0.0

0.7

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Fetal skeletal variations

For all test groups, skeletal variations of different bone structures were observed, with or

without effects on corresponding cartilages. The observed skeletal variations were related to

several parts of fetal skeletons and appeared without a relation to dose. The

overall incidences of skeletal variations were comparable to the historical control data.

Tab. 9: Total fetal skeletal varations

Test group0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

LitterFetuses

N N

129

131

119

143

Fetal incidence

N (%)

124 (96)

126 (96)

117 (98)

140 (98)

Litter incidence

N (%)

23 (100)

25 (100)

23 (100)

25 (100)

Affectedfetuses/litter

Mean%

96.5

98.0

97.9

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

For a better overview, all skeletal variations with statistically significant differences between

the control and any treated group were compiled in the table below. All

incidences were expressed on a fetus per litter basis.

Tab. 10: Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

Finding

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

HCD

Mean % (range)

Incomplete ossification of supraoccipital; unchanged cartilage

20.2

16.8

34.2*

18.9

32.2

(14.5 – 63.5)

Misshapen sacral vertebra

1.3

3.2

0.9

5.5*

4.1

(0.7 – 9.8)

Unossified sternebra; unchanged cartilage

2.1

2.3

3.4

9.1*

4.3

(0.0 – 9.7)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent

* = p ≤0.05 (Wilcoxon-test [one-sided]) ** = p ≤0.01 (Wilcoxon-test [one-sided])

The increased incidences of skeletal variations were not

related to the dose or they were clearly inside the historical control range. Therefore, they are

not considered as treatment-related and adverse.

Fetal skeletal unclassified cartilage observations

Additionally, some isolated cartilage findings without impact on the respective bony structures,

which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the ribs and

the sternum and did not show any relation to dosing.

Tab. 11: Total unclassified cartilage observations

Test group0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

LitterFetuses

N N

129

131

119

143

Fetal incidence

N (%)

89 (69)

79 (60)

73 (61)

102 (71)

Litter incidence

N (%)

23 (100)

24 (96)

22 (96)

25 (100)

Affectedfetuses/litter

Mean%

70.1

60.9

59.6

70.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Assessment of all fetal external, soft tissue and skeletal observations

There were noted external, soft tissue and skeletal malformations in all test groups (0, 100, 300 and 1000 mg/kg bw/d).

Three fetuses carried more than one malformation. Female control fetus No. 15-03 (0 mg/kg

bw/d) had multiple external malformations (i.e. gastroschisis and ectrodactyly) associated with

multiple skeletal malformations (i.e. absent forepaw phalanx and severely malformed sternum).

For female control fetus No. 25-14 hydronephrosis and hydroureter were recorded, while female

low-dose fetus No. 33-10 (100 mg/kg bw/d) had a bipartite basisphenoid, misshapen

presphenoidal and a severely malformed vertebral column and/or ribs (in the region of thoracic

vertebrae). The body weight of this fetus (3.2 g) was lower compared to its group mean value

(females of test group 1: 3.5 g). Further malformations, i.e. umbilical hernia, situs inversus,

interrupted spinal cord, anophthalmia, absent subclavian and malpositioned and bipartite

sternebrae were observed in individual fetuses, unrelated to the dose and, except ‘interrupted

spinal cord’ (control fetus), all of them can be found in the historical control data.

All these findings were single cases, no ontogenetic pattern is recognizable for all these individual

malformations nor was there any cluster of any of these individual malformations seen

in the other offspring of these test groups. They also do neither form a pattern or syndrome

with other minor anomalies which may raise toxicological concern. There is no evidence for

any association of these scattered findings with the treatment.

Tab. 12: Total fetal malformations

Test group0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

LitterFetuses

251

248

229

273

Fetalincidence

N (%)

5 (2.0)

1 (0.4)

4 (1.5)

Litter

incidence

N (%)

5 (22)

1 (4.0)

1 (4.3)

4 (16)

Affectedfetuses/litter

Mean%

2.0

0.4

1.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

External variations did not occur in any of the fetuses in this study. Three soft tissue variations

and a range of skeletal variations were noted

in all test groups including the controls. None of the total incidences showed a relation to dose. The individual variations were equally distributed about the different test

groups, if normal biological variation is taken into account, and can be found in the historical

control data at a comparable frequency.

Tab. 13: Total fetal variations

Test group0

0 mg/kgbw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

LitterFetuses

251

248

229

273

Fetalincidence

N (%)

127 (51)

128 (52)

119 (52)

144 (53)

Litterincidence

N (%)

23 (100)

25 (100)

23 (100)

25 (100)

Affectedfetuses/litter

Mean%

50.8

51.6

52.3

52.6

mg/kg bw/d = milligram per kilogram body weight per day; N= number; % = per cent

No unclassified external and unclassified soft tissue observations were recorded for any of the

fetuses in this study. A spontaneous origin is assumed for the unclassified skeletal cartilage

observations which were observed in several fetuses of all test groups

(0, 100, 300 and 1000 mg/kg bw/d). The distribution and type of these findings do not suggest

any relation to treatment.

Finally, fetal examinations revealed that there is no effect of the compound on the respective

morphological structures up to the highest dose tested (1000 mg/kg bw/d).

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 1000 mg/kg bw/d caused neither evidence of maternal nor fetal developmental toxicity.
In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested dose of 1000 mg/kg bw/d.

Executive summary:

The test substance was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered as an oily preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (corn oil) in parallel. A standard dose volume of 4 mL/kg body weight was used for each test group.

At terminal sacrifice on GD 20, 23-25 females per group had implantation sites. The analytical values of the low-dose samples (100 mg/kg bw/d) were above the expected range of 90% to 110% of the nominal concentrations. The mean value of around 138% of the nominal concentration corresponds to an actual low-dose of 138 mg/kg bw/d. For the mid- and high dose, the correctness of the prepared concentrations was shown. This did not affect the validity of the study. In the following report, the target doses were used.

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

On GD 20, all surviving females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

The stability of the test substance preparations over a period of 7 days at room temperature was demonstrated. The correctness of the prepared concentrations was shown (mid- and high-dose). Concentration control analysis of the low-dose: the three analytical samples resulted in a mean value of around 138% of the nominal concentration.

The following test substance-related adverse effects/findings were noted:

Test group 3 (1000 mg/kg bw/d):

• No test substance-related adverse effects on dams, gestational parameters or fetuses

Test group 2 (300 mg/kg bw/d):

• No test substance-related adverse effects on dams, gestational parameters or fetuses

Test group 1 (100 mg/kg bw/d):

• No test substance-related adverse effects on dams, gestational parameters or fetuses

In conclusion, the NOAEL was assessed as 1000 mg/kg bw/day for maternal and fetal developmental toxicity.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 2019 - March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 1998

Deviations:

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Jun 2018

Deviations:

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2018

Deviations:

Qualifier:

according to guideline

Guideline:

other: JMAFF Guideline 2-1-18

Version / remarks:

Nov 2000

Deviations:

GLP compliance:

yes

Limit test:

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: BASF SE, Ludwigshafen, Germany
- Lot No.of test material: D688149955
- Purity: 99.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: An emulsion was able to be prepared at 250 mg/ml. The test substance was found to be stable at concentrations ranging from 2.5 mg/ml to 250 mg/ml for up to 7 days.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Covance Research Products, Inc. (CRP), Greenfield, Indiana
- Age at study initiation: sexually mature adults
- Weight at study initiation: 2800-3200 g
- Housing: individually
- Diet: 75 g - 150 g
- Water: ad libitum
- Acclimation period: at least four days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): average 20; range 16-22
- Humidity (%): 40-60
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12 (6 am to 6 pm; illumination)

IN-LIFE DATES: From: 2/18/2019 (Test substance administration ) To: 3/20/2019

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose (METHOCEL)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Test material was prepared by mixing with 0.5% methylcellulose in ultrapure water+Cremophor EL (10 mg/100 ml) at concentrations of 0, 25, 75, or 250 mg/ml.
- Administration volume: 4 ml/kg bw

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance was determined to be insoluble in all vehicles tested other than the prepared emulsion.
- Concentration in vehicle: 0.5% methylcellulose in ultrapure water + Cremophor EL (10 mg/100 ml)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose confirmation analyses of all dose levels plus control were determined preexposure. The homogeneity of the low-dose and the high-dose test emulsions was determined concurrent with dose confirmation. The method used for analyzing the test material in 0.5% methylcellulose (METHOCEL™) in ultrapure water +
Cremophor EL (10mg/100mL) was a solvent extraction method followed by gas chromatography-flame ionization detector (GCFID).

Details on mating procedure:

Each sexually mature virgin female was naturally mated with one buck of the same strain at Covence Research Products (CRP). The observed day of breeding was considered GD 0. GD 0 body weights and records of mating pairs were provided by CRP and maintained in the study record.
Rabbits arrived in laboratory on GD 1 or 2.

Duration of treatment / exposure:

20 days

Frequency of treatment:

daily

Duration of test:

GD 7 - 27

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:

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected on the basis of the developmental
toxicity probe study. Due to the highly variable data within each dose group (excluding the GD 7-10 time period), and a lack of a dose response in clinical observations, body weights, body weight gains, and feed consumption in the probe study, these findings were not used to limit the high dose for this study. The high-dose of 1000 mg/kg/day represented a limit dose as defined in the Organization for Economic Co-operation and Development Test Guideline 414 (OECD 414 Prenatal Developmental Toxicity Study). The lower dose levels were selected to provide dose response data for any toxicity that may have been observed among the high dose group rabbits.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Bodz weights were recorded on GD 0 (by the supplier), daily during the dosing period, and on GD 28 (terminal). Statistical analysis of body weights was performed using data collected on GD 0, 7, 10, 13, 16, 20, 24, and 28. Statistical analysis of body weight gains was conducted for the

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Consumption was recorded and analyzed from GD 4-28

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Organs examined: stomach, liver, kidney, uterus

OTHER:
- Microscopic examination of tissues was not conducted.

Ovaries and uterine content:

Fetal examinations:

- External examinations: Yes with the exception of one litter from Dam #98 of the 1000 mg/kg/day group that was inadvertently not recorded)
- Soft tissue examinations: Yes (with the exception of fetus #5 from Dam #18 of the control group that was inadvertently not recorded)
- Skeletal examinations: Yes
- Head examinations: Yes: [half per litter] with the exception of one litter that was inadvertently not recorded (fetuses from Dam # 14 of the control group).

Statistics:

- Bartlett's test (alpha = 0.01) for Maternal body weights, maternal body weight gains, organ weights (absolute and relative with the exception of only absolute weight for gravid uterus), fetal body weights and feed consumption
- parametric or nonparametric ANOVA. If ANOVA was significant at alpha = 0.05 analysis by Dunnett's test or the Wilcoxon Rank-Sum test with Bonferroni's correction was performed
- Censored Wilcoxon test with Bonferroni's correction for Frequency of pre- and post-implantation loss (calculations shown below), and fetal alterations (if any)
- Nonparametric ANOVA (alpha = 0.05) followed by the Wilcoxon Rank-Sum test (alpha = 0.05) with Bonferroni's correction for number of corpora lutea, implantations, and litter size
- Fisher exact probability test (alpha = 0.05) with Bonferroni's correction for pregnancy rates

Indices:

Pre-and post-implantation loss

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There was an increased incidence of transient decreased feces in the 1000 mg/kg/day dose group compared to controls. This finding was associated with decreases in feed consumption.
For details, see attached table(s) in PDF (see attached background material)

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 1000 mg/kg/day: mean body weight loss of 38.3g, from GD 7-10, and an overall body weight gain decrease of 22.5% from GD 7-28 compared to controls
- 300 mg/kg/day: decrease of 68.4% in body weight gain from GD 7-10 (non-adverse). Body weight gains over entire treatment period (GD 7-28) comparable to controls and thus not adverse
For details, see attached table(s) in PDF (see attached background material)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- 1000 mg/kg/day: decrease in food consumption from GD 7-14 ranging from 6.8%-22.1%
For details, see attached table(s) in PDF (see attached background material)

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Scheduled cesarean section

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

- no effects on numbers of corpora lutea
- no effects on gravid uterine weights
For details, see attached table(s) in PDF (see attached background material)

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: evidence of systemic toxicity at 1000 mg/kg bw/day including decreased body weight gain and feed consumption

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related skeletal alterations in any dose group.
- statistically-identified decrease in the incidence of delayed ossification (DO) of the hyoid in the low and mid dose groups compared to control. No toxicological relevance since incidences were lower than control
- incidental malformations: missing ribs, fused thoracic rib, fused thoracic centra, thoracic hemicentric centra, thoracic hemivertebra and fused lumbar centra bearing no relationship to treatment
- incidental variations: DO pubis, calloused ribs, fused sternebrae, irregular pattern of ossification sternebrae, extra site of ossification sternebrae, DO sternebrae, crooked hyoid, and DO hyoid bearing no relationship to treatment
Given that these observations occurred in the control group, occurred at low frequencies, and/or lacked a dose response, these observations were considered spurious and unrelated to treatment.
For details, see attached table(s) in PDF (see attached background material)

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related or statistically-identified visceral alterations in any dose group.
Incidental findings bearing no relationship to treatment included the malformations of enlarged aorta, ventricular septal wall defect, persistent truncus arteriosus, misshapen heart, and missing gall bladder, and variations of missing caudal lung lobe, liver cyst, right-sided esophagus, hemorrhage thymus, supernumerary hepatic lobule, retrocaval ureter, paraovarian cyst, and paratesticular cyst. Given that these observations occurred in the control group, occurred at low incidences, and/or lacked a dose response, these observations were considered spurious and unrelated to treatment.
For details, see attached table(s) in PDF (see attached background material)

Other effects:

no effects observed

Description (incidence and severity):

- no craniofacial alterations in any dose group
For details, see attached table(s) in PDF (see attached background material)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

effects observed, non-treatment-related

Developmental effects observed:

RESULTS AND DISCUSSION

Analytical

The dose confirmation and homogeneity report is provided in Appendix A. Analysis of

all dosing emulsions from the first mix revealed mean acceptable concentrations of npropyl

acetate ranging from 89.8% to 97.6% of targeted concentrations.

Analysis of aliquots for the low- and high-dose emulsions indicated that the test material

was homogeneously distributed based on relative standard deviations of 9.15 and 3.43%,

respectively.

In-Life Observations

Clinical and cage-side observations are summarized in Table 2 and individual data are

reported in Appendix Table 1.

There was an increased incidence of transient decreased feces in the 1000 mg/kg/day

dose group compared to controls. This finding was associated with decreases in feed

consumption. All other clinical observations were considered unrelated to treatment due

to the low incidence.

Body Weights/Body Weight Gains

Mean body weight and body weight gain data for pregnant rabbits are summarized in

Tables 3 and 4, individual data for all females are reported in Appendix Tables 2 and 3.

Rabbits administered 1000 mg/kg/day had a treatment-related mean body weight loss of

38.3g, from GD 7-10, and an overall body weight gain decrease of 22.5% from GD 7-28

compared to controls (Text Table 3). Rabbits administered 300 mg/kg/day had a

treatment-related decrease of 68.4% in body weight gain from GD 7-10 compared to

controls; however, over the entire treatment period (GD 7-28), body weight gains were

similar to controls and were therefore considered non-adverse. Body weights for all

treated groups were similar to controls and there were no treatment-related body weight

gain changes in the 100 mg/kg/day dose group compared to controls.

Text Table 3. Selected Mean Body Weight Gains (g)

Dose (mg/kg/day)	Gestation Day
Dose (mg/kg/day)	7-10 Gain	10-13 Gain	7-28 Gain
0	17.4	53.5	307.9
100	18.2	40.4	331.8
300	5.5	60.5	303.2
1000	-38.3*	54.0	238.5

Boldvalues were considered treatment-related effects.

*Statistically Different From Control Mean By Wilcoxon's Test, Alpha=0.05.

Feed Consumption

Mean feed consumption data for pregnant rabbits are summarized in Table 5, individual

data for all females are reported in Appendix Table 4.

Rabbits administered 1000 mg/kg/day had treatment-related decreases in feed

consumption from GD 7-14 compared to controls ranging from 6.8-22.1% (Text Table

4). During the last two weeks of gestation (GD 14-28), the feed consumption amongst all

the dose groups was more variable. However, the feed consumption in the

1000 mg/kg/day dose group was consistently lower than controls across all gestation

days. There were no treatment-related differences in the amount of feed consumed by the

100 or 300 mg/kg/day dose groups when compared to controls.

Text Table 4. Selected Feed Consumption Intervals (g)

Dose (mg/kg/day)	Gestation Day
Dose (mg/kg/day)	7-8	8-9	9-10	10-11	11-12	12-13	13-14
0	134.6	134.4	132.6	132.7	129.9	115.5	116.6
100	133.4	134.0	134.6	131.4	127.8	121.1	117.9
300	131.4	128.4	130.6	120.6	123.5	117.2	110.4
1000	104.9	111.5*	114.4*	113.4*	110.3	107.6	103.8

Boldvalues were considered treatment-related effects.

* Statistically Different From Control Mean By Dunnett's Test, Alpha=0.05.

Anatomic Pathology

Organ Weights

Terminal body, liver, and kidney weight data for pregnant rabbits are presented in

Table 6, individual data are reported in Appendix Table 5.

There were no treatment-related or statistically-identified differences in any of the

measured parameters for any treated groups when compared to controls.

Gross Pathology

A summary of gross pathologic observations are presented in Table 7, and

individual data are reported in Appendix Table 6.

There were no treatment-related gross pathologic observations.

Reproductive Parameters

Reproductive parameters measured at necropsy are summarized in Table 8 and individual

data are reported in Appendix Table 7.

There were no treatment-related or statistically-identified effects on pregnancy rates,

resorption rates, litter size, numbers of corpora lutea or implantations, percent

preimplantation loss, percent post-implantation loss, fetal sex ratios, fetal body weights or

gravid uterine weights at any dose level.

Fetal Examination Summary

The incidence of external, visceral, craniofacial, and skeletal variations and

malformations observed among fetal rabbits is summarized in Table 9, while individual

litter data are reported in Appendix Table 8. Malformation historical control data are

presented in Appendix B.

Compared to controls, there were no treatment-related differences in the incidence of any

fetal alteration in any of the treated groups. The small number of alterations observed in

fetuses from dams administered n-propyl acetate either occurred at low frequencies

and/or were not dose related. Details of these findings are described below.

External Examination

There were no alterations in any dose group.

Visceral Examination

There were no treatment-related or statistically-identified visceral alterations in any

dose group. Incidental findings bearing no relationship to treatment included the

malformations of enlarged aorta, ventricular septal wall defect, persistent truncus

arteriosus, misshapen heart, and missing gall bladder, and variations of missing

caudal lung lobe, liver cyst, right-sided esophagus, hemorrhage thymus,

supernumerary hepatic lobule, retrocaval ureter, paraovarian cyst, and paratesticular

cyst. Given that these observations occurred in the control group, occurred at low

incidences, and/or lacked a dose response, these observations were considered

spurious and unrelated to treatment.

Craniofacial Examination

There were no alterations in any dose group.

Skeletal Examination

There were no treatment-related skeletal alterations in any dose group. There was a

statistically-identified decrease in the incidence of delayed ossification (DO) of the

hyoid in the low and mid dose groups compared to control. This finding has no

toxicological significance as the incidences were lower than controls. Other

incidental findings bearing no relationship to treatment included the malformations

missing ribs, fused thoracic rib, fused thoracic centra, thoracic hemicentric centra,

thoracic hemivertebra and fused lumbar centra. Incidental variation findings

bearing no relationship to treatment were DO pubis, calloused ribs, fused

sternebrae, irregular pattern of ossification sternebrae, extra site of ossification

sternebrae, DO sternebrae, crooked hyoid, and DO hyoid. Given that these

observations occurred in the control group, occurred at low frequencies, and/or

lacked a dose response, these observations were considered spurious and unrelated

to treatment.

Conclusions:

Treatment-related clinical observations were limited to an increased incidence of transient decreased feces in the 1000 mg/kg/day dose group compared to controls.
Rabbits administered 1000 mg/kg/day had a treatment-related mean body weight loss from GD 7-10 with concomitant decreases in feed consumption. This resulted in an overall 22.5% decrease in body weight gain from GD 7-28 compared to controls. Rabbits administered 300 mg/kg/day had a treatment-related decrease in body weight gain from GD 7-10 compared to controls, however over the entire treatment period (GD 7-28), body weight gains were similar tocontrols and were therefore considered non-adverse.
There were no treatment-related differences in gross pathologic observations or maternal kidney or liver weights for any of the treated groups when compared to controls.
There were no indications of embryo/fetal toxicity or teratogenicity at any dose level.

Under the conditions of this study and based on evidence of systemic toxicity at the high dose level including decreased body weight gain and feed consumption, the no-observedadverse-effect level (NOAEL) for maternal toxicity was 300 mg/kg/day. The no-observed-effect level (NOEL) for developmental toxicity was 1000 mg/kg/day, the highest dose tested.

Executive summary:

The purpose of this study was to evaluate the potential maternal and developmental toxicity of the test substance in New Zealand White (NZW) rabbits following repeated oral gavage administration. Groups of 24 time-mated female rabbits were administered n-propyl acetate in 0.5% methylcellulose (METHOCEL™) in ultrapure water + Cremophor EL (10mg/100mL) via gavage at dose levels of 0, 100, 300, or 1000 milligrams per kilogram body weight per day (mg/kg/day) on gestation day (GD) 7-27. In-life parameters evaluated for all groups included: clinical observations, body weight, body weight gain, and feed consumption.

On GD 28 all surviving rabbits were euthanized and examined for gross pathologic alterations. Liver, kidneys, and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions, and live/dead fetuses. Fetuses were weighed, sexed, and examined for external and visceral alterations. Also, the heads were examined for craniofacial alterations by serial sectioning in approximately one half of the fetuses in each litter, while skeletal examinations were performed on all fetuses.

Rabbits administered 300 mg/kg/day had a treatment-related decrease in body weight gain from GD 7-10 compared to controls, however over the entire treatment period (GD 7-28), body weight gains were similar to controls and were therefore considered non-adverse.

There were no treatment-related differences in gross pathologic observations or maternal kidney or liver weights for any of the treated groups when compared to controls. There were no indications of embryo/fetal toxicity or teratogenicity at any dose level. Under the conditions of this study and based on evidence of systemic toxicity at the high dose level including decreased body weight gain and feed consumption, the no observed-adverse-effect level (NOAEL) for maternal toxicity was 300 mg/kg/day. The no-observed-effect level (NOEL) for developmental toxicity was 1000 mg/kg/day, the highest dose tested.

Effect on developmental toxicity: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 1 000 mg/kg bw/day
Study duration:: subacute
Species:: other: rat and rabbit

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:: no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:: no study available

Additional information

OECD 414 in rats:

The test substance propyl acetate was tested for its prenatal developmental toxicity in Wistar rats. The study was conducted according to OECD 414 guideline and GLP. The test substance was administered as an oily preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (corn oil) in parallel. A standard dose volume of 4 mL/kg body weight was used for each test group.

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

The following test substance-related adverse effects/findings were noted:

Test group 3 (1000 mg/kg bw/d):

• No test substance-related adverse effects on dams, gestational parameters or fetuses

Test group 2 (300 mg/kg bw/d):

• No test substance-related adverse effects on dams, gestational parameters or fetuses

Test group 1 (100 mg/kg bw/d):

• No test substance-related adverse effects on dams, gestational parameters or fetuses

In conclusion, the NOAEL was assessed as 1000 mg/kg bw/day for maternal and fetal developmental toxicity.

OECD 414 in rabbits:

A GLP-conform study according to OECD 414 was conducted in rabbits (2019). The purpose of this study was to evaluate the potential maternal and developmental toxicity of the test substance in New Zealand White (NZW) rabbits following repeated oral gavage administration. Groups of 24 time-mated female rabbits were administered n-propyl acetate in 0.5% methylcellulose (METHOCEL™) in ultrapure water + Cremophor EL (10mg/100mL) via gavage at dose levels of 0, 100, 300, or 1000 milligrams per kilogram body weight per day (mg/kg/day) on gestation day (GD) 7-27. In-life parameters evaluated for all groups included: clinical observations, body weight, body weight gain, and feed consumption.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation 1272/2008. Based on the criteria laid down in Regulation (EC) No. 1272/2008, as amended for the second time in Directive EC 286/2011, classification for reproduction toxicity is not warranted.

Additional information

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Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

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Diss Factsheets

Propyl acetate

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Link to relevant study records

Reference

Effect on fertility: via oral route

Effect on fertility: via inhalation route

Effect on fertility: via dermal route

Additional information

Effects on developmental toxicity

Description of key information

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

Effect on developmental toxicity: via oral route

Effect on developmental toxicity: via inhalation route

Effect on developmental toxicity: via dermal route

Additional information

Justification for classification or non-classification

Additional information

Referenceopen all close all