Condensation products of Dihydrofuran-2,5-dione, mono-alkenyl derivs. with polyethylene amine and acetic anhydride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Fertility was measured with EXP0700332 at 100, 300 and 1000 mg/kg.day in the OECD 421 and 416 guidelines. There was no impact on fertility with a NOEL > 1000 mg/kg/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 June 2015 to 26 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

The test and control items were administered to the appropriate F0 and F1 animals by once daily oral gavage for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the resultant litters had reached Day 21 of lactation. The F1 pups selected to form the F1 adult generation commenced dosing within 1 week of weaning (at ca 4 weeks of age).
The volume for each animal was based on the most recent body weight measurement except during late gestation; from Day 16 until parturition was completed, the dose volume was determined by the weight of the animal on Day 16 of gestation (see Appendix 1 for details of deviation). The doses were given using a syringe with attached gavage cannula.
For both generations, females were not dosed if they were found to be littering at the time that dose administration was scheduled.

The Han Wistar rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies and the Test Facility has sufficient historical control data in this species and strain.
The total number of animals used in this study was considered the minimum required to properly characterise the effects of the test item. This study was designed such that it did not require an unnecessary number of animals to accomplish its objectives.
The oral gavage route of administration was selected for this study as this route was defined by the Sponsor as a possible route of human exposure.

The dose levels were agreed with the Sponsor following evaluation of a previous twenty-eight day repeated dose oral (gavage) toxicity study in the rat (OECD Guideline 407), a reproduction / developmental toxicity screening of study oral gavage in the rat (OECD Guideline 421; Charles River Study No. 496381) and a Prenatal Developmental Toxicity Study in the rat (OECD Guideline 414; Charles River Study No. 497233) performed on behalf of the Sponsor.

F0 and F1 male rats were euthanised once the majority of females reached the end of lactation. F0 and F1 females were euthanisd at or shortly after weaning (ca. Day 21 of lactation). Pups not selected for post-weaning analysis were killed at the same time as thier mother. Animals 10 days of age or more were killed by exposure to carbon dioxide followed by exsanguination. Animals less than 10 days of age were killed by intra-peritoneal injection of sodium pentobarbitone followed by exsanguination. Animals were euthanised rotating across dose groups such that similar numbers of animals from each group, including controls were necropsied throughout each day of necropsy.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch No.of test material: E00031-541
- Expiration date of the lot/batch: 01 November 2016
- Purity test date: > 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature in the dark

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Han Wistar rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies and the Test Facility has sufficient historical control data in this species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd, Margate, Kent, UK.
- Age at study initiation: (P) Males: 7-8 weeks; females: 6 - 7 weeks; (F1) approximately 4 wks
- Weight at study initiation: (P) Males: 164-238 g; Females: 114-164 g; (F1) Males: 248-256 g; Females: 173-181 g
- Housing: All animals were initially housed 2 or 3 per cage in appropriately sized polycarbonate cages with stainless steel grid tops and bottoms. A few days prior to pairing for mating, the males were transferred to individual cages which females were then transferred to for mating. Males were re-caged to their original cage-mates.
- Diet (e.g. ad libitum): ad libitum, except during designated procedures
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-25 °C
- Humidity (%): 43-69%
- Air changes (per hr): At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark
IN-LIFE DATES: From: 08 June 2015 To: 04 December 2015

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

SDS VRF-1 breeder diet was provided ad libitum throughout the study, except during designated procedures. The feed was analysed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are retained at the Test Facility.There were no known contaminants in the feed that would interfere with the objectives of the study.

VEHICLE
- Volume dosage: 4 mL/kg
The control formulation, Arachis Oil BP, was stored in a refrigerator set to maintain 4°C, and dispensed daily. The control formulation was removed from the refrigerator and stirred continuously from at least 30 minutes prior to dosing until dosing was completed each day. Any residual volumes were discarded.

Details on mating procedure:

A few days prior to the initiation of mating, the males were separated into individual solid bottomed cages. Pairing was on a 1:1 or 1:2 (male: female) basis in ascending numerical order. Each female was transferred to the cage of its appropriate co-group male during the evening of Day 71 of dosing (where Day 1 was first day of dosing) from 19.00 onwards.
Vaginal lavages were taken each morning until mating occurred and the stage of oestrus observed in each vaginal lavage was recorded. The day of detection of a copulatory plug in situ and/or of sperm in the lavage was designated Day 0 of gestation.
The pairing period was a maximum of 14 nights. If evidence of mating was not observed by the end of the pairing period, the female was separated from the male during the morning following the last night of pairing and treated as if it had mated during that night.
For each female the time taken to show a positive mating sign and the number of failed opportunities to mate (oestruses passed without a sign of mating) were evaluated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate top, middle and bottom (duplicate middle only for control and Group 3) samples (1 mL) for each sampling time point were sent to the analytical laboratory. Triplicate top, middle, and bottom samples (triplicate middle only for control and Group 3) were maintained at the Test Facility as backup samples. Samples were kept in a refrigerator set to maintain 4ºC. After acceptance of the analytical results, backup samples were discarded.
For concentration, the criterion for acceptability was a mean sample concentration result within or equal to ± 10% of theoretical concentration, with individual samples within ± 20%. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentrations of = 10% for each group.

Duration of treatment / exposure:

F0 animals were dosed for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the (F1) litter had reached Day 21 of lactation. From each dose group, up to 24 males and 24 females were retained for post weaning assessments. These animals were dosed for 10 weeks after weaning, and then throughout mating, gestation and lactation until termination after the (F2) litter had reached Day 21 of lactation.

Frequency of treatment:

daily

Details on study schedule:

F1 dosing commenced within 1 week of weaning (ca. 4 weeks of age). From each group, 24 males and females were selected for post-weaning assessments by selecting up to one male and one female from each litter. Pups were selected by median weight, removed from their mother on Day 21 of lactation, and individually identified and housed in their new cage. These animals were dosed for 10 weeks after weaning, throughout mating, gestation and lactation until termination after the F2 litter had reached Day 21 of lactation. Pups not selected for post-weaning assessments remained with their mother until sacrifice.
Sexual maturation assessments commenced at 28 and 35 days of age for females and males respectively. Females were examined for vaginal opening and males were examined for balano-preputial separation. Body weights were recorded on the day of sexual maturation.

Dose / conc.:

0 mg/kg bw/day

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:

F0: 28 animals per sex per dose; F1: 24 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The test and control items were administered to the appropriate F0 and F1 animals by once daily oral gavage for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the resultant litters had reached Day 21 of lactation. The F1 pups selected to form the F1 adult generation commenced dosing within 1 week of weaning (at ca 4 weeks of age). The volume for each animal was based on the most recent body weight measurement except during late gestation; from Day 16 until parturition was completed, the dose volume was determined by the weight of the animal on Day 16 of gestation. The doses were given using a syringe with attached gavage cannula. For both generations, females were not dosed if they were found to be littering at the time that dose administration was scheduled.

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- All animals were examined at least once a day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Amimals were removed from the cage and examined weekly commencing Week -1 throughout the study.

BODY WEIGHT: Yes
- Male body weights were recorded once during pre-treatment and weekly throughout the dosing period. F0 female body weights were recorded once during pre-treatment, weekly until pairing for mating, on Days 0 (the day of detection of positive mating sign), 7, 14, 16 and 20 of gestation and on Days 1 (day of birth of litter), 7, 14 and 21 of lactation. A weekly regimen was applied to females that had not shown a positive mating sign, until parturition or termination. Retained F1 animals body weights were recorded weekly commencing on a suitable day within one week of weaning of the majority of litters until termination for males or until pairing for mating for the females; during gestation and lactation, weighing of F1 females followed the same regimen as F0 females.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was quantitatively measured weekly commencing at the start of Week -1 (F0 animals) or from a suitable day within one week of weaning of the majority of animals (F1 animals) until pairing for mating. Males – Weekly measurements recommenced after the mating period. Mated Females – Measured over Days 0-7, 7-14 and 14-20 of gestation, and over Days 0-7, 7-14 and 14-21 of lactation. A weekly regimen was applied to females that had not shown a positive mating sign, until parturition or termination.

Oestrous cyclicity (parental animals):

Over a 2-week period prior to the initiation of mating and throughout the mating period, vaginal lavages were taken each morning and the stages of oestrous observed was recorded.

Sperm parameters (parental animals):

From the first 10 males/group from both the F0 and F1 generations, the right cauda epididymis was placed into Medium 199 (as per SOP/PAT/069) and the sperm were allowed to “swim out” into the medium. An appropriate dilution of the sperm suspension was prepared and examined using a Hamilton Thorne sperm motility analyser.
The remaining portion of the cauda epididymis was minced and suspended. Dilutions of this sperm suspension were counted using a haemocytometer to obtain a total sperm count which was expressed per sample. From all samples of the sperm suspensions described in the preceding paragraph, a sperm smear was prepared and stained with eosin Y solution. From the Group 1 and 4 animals (both generations), at least two hundred sperm per animal were evaluated for morphological abnormalities using criteria described by Wyrobek and Bruce (1975).
One testis was decapsulated and homogenised. The homogenate was sonicated to reduce tissue debris, if required. The number of homogenisation resistant spermatids in dilutions of this suspension were counted using a haemocytometer to obtain a total spermatid count which was expressed per testis and per gram of testis.

Litter observations:

The day of birth of the litter (day on which first pups were born) was designated Day 0 of lactation. The duration of gestation in days was calculated. The numbers of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were counted and examined for the presence of milk in the stomach and for any externally visible abnormalities daily up to at least Day 21 of lactation. These pups were weighed en masse, sexes separately, on Days 1, 4, 7 and 14 of lactation. On Day 21 of lactation they were weighed individually. Deficiencies in maternal care were recorded: inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding.

Postmortem examinations (parental animals):

SACRIFICE
- Males - once the majority of females reached the end of lactation.
- Females - At or shortly after weaning (ca. Day 21 of lactation).
Animals 10 days of age or more were killed by exposure to carbon dioxide followed by exsanguination. Animals less than 10 days of age were killed by intra-peritoneal injection of sodium pentobarbitone followed by exsanguination. Animals were euthanized rotating across dose groups such that similar numbers of animals from each group, including controls were necropsied throughout each day of necropsy.

GROSS NECROPSY
- Animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. The reproductive tract of all adult females was examined for signs of implantation, the number of implantation sites were recorded. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathological evaluation was performed by a board-certified veterinary pathologist with training and experience in laboratory animal pathology. A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-specificity or stage-specificity of testicular findings were noted. The examination of the ovaries included quantification of the primordial and growing oocytes, and the confirmation of the presence or absence of the corpora lutea.

The following organs were weighed at necropsy: Brain, Epididymis, Adrenal gland, Pituitary gland, Prostate gland, Thyroid gland, Kidney, Liver, Ovary, Spleen, Testis and Uterus. Organ weights were not recorded for animals found dead or euthanised in poor condition or in extremis. Paired organs were weighed separately but have been reported together. Terminal body weights were used for organ weight analysis.

Postmortem examinations (offspring):

Termination occured onced the litter had reached Day 21 of lactation.
From each litter, two male and two female pups (where they were available) were necropsied. This consisted of external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of any grossly abnormal tissues were preserved in 10% formalin or other appropriate fixative. From one of the two pups of each sex (where they were available), the weights of the brain, spleen and thymus were recorded (as was as a terminal body weight), and these organs were preserved. Representative samples of any abnormal tissues from any of the pups were also preserved. The carcasses were then discarded.
Where 2 pups of either sex were not available for necropsy, or 1 pup of each sex were not available for organ weight collection, additional pups of the opposite sex were necropsied/weighed such that 4 animals per litter were necropsied and 2 animals per litter had organs weighed as far as was possible.
The remaining pups in each litter were checked for externally visible abnormalities at the time of killing. Any found to have such an abnormality was necropsied as described in the preceding paragraph. The remaining carcasses were discarded.

Statistics:

Tests were applied to determine the statistical significance of observed differences between the control group and groups receiving test item. Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group. Body weight and food consumption were analysed for homogeneity of variance using the ‘F-Max’ test. If the group variance appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F-protected LSD method via Student’s t-test ie pairwise comparisons were made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (Via z tests, the non-parametric equivalent of Student’s t test). Organ weight data was analysed as above, and by analysis of covariance (ANCOVA) using terminal body weight as the covariate.

Reproductive indices:

For each group the fertility index (male and female) and gestation indices were calculated. For each litter and group the birth indices, live birth indices, viability indices, lactation indices and the overall survival indices were calculated.

Offspring viability indices:

For each group, the following reproductive indices were calculated: Fertility Index (male and female) and gestation index.
For each litter and group: birth index, live birth index, viability index, lactation index and overall survival index.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Slow respiration and subdued behaviour. A swelling was observed in the right lower ventral abdomen which was noted as a firm brown subcutaneous mass involving the nipple at necropsy; grossly the nipple itself was reddened and damaged with pale material at the centre; inflammation and necrosis of the mammary gland was noted at histopathological examination.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no test item-related deaths - there were a number of adult animals found dead or prematurely euthanised however the incidence and circumstances surrounding these deaths indicated no relationship to the test item.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

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

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOEL

Effect level:

ca. 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

ophthalmological examination

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were a number of adult animals found dead or prematurely euthanised however the incidence and circumstances surrounding these deaths indicated no relationship to the test item.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry 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:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Pertinent pathology findings in relation to animals that failed to mate or produce a live litter are discussed in the pathology report; in none of these cases was the test item considered to be a factor.

Key result

Dose descriptor:

NOEL

Effect level:

ca. 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

ophthalmological examination

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

In both generations, there were a number of pups found dead or prematurely euthanised, however the incidence and circumstances surrounding the deaths indicated no relationship to the test item.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

ca. 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

sexual maturation

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

ophthalmological examination

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

developmental neurotoxicity

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

In both generations, there were a number of pups found dead or prematurely euthanised, however the incidence and circumstances surrounding the deaths indicated no relationship to the test item.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOEL

Generation:

F2

Effect level:

ca. 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

developmental neurotoxicity

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

In conclusion, once daily oral gavage administration of EXP0700332 to rats at dose levels of up to 1000 mg/kg/day from 10 weeks prior to mating, and then throughout mating, gestation and lactation was well tolerated and had no effect upon the reproductive performance of either the initial (F0) generation or the subsequent (F1) generation. Therefore, under the conditions of this study the no observed effect level (NOEL) was considered to be 1000 mg/kg/day.

Executive summary:

This study was designed to provide general information concerning the effects of EXP0700332 on the reproductive performance of rats when administered once daily via oral gavage over two consecutive generations. F0 animals were dosed for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the (F1) litter had reached Day 21 of lactation. From each dose group, up to 24 males and 24 females were retained for post weaning assessments. These animals were dosed for 10 weeks after weaning, and then throughout mating, gestation and lactation until termination after the (F2) litter had reached Day 21 of lactation. Both F0 and F1 animals were dosed at 0, 100, 300 and 1000 mg/kg/day. The following parameters and end points were monitored/evaluated in this study: clinical signs, body weights, body weight gains, food consumption, oestrous cycles, mating performance, fertility, pregnancy performance (including difficulty or prolongation of parturition), deficiencies in maternal care, offspring growth and survival, assessment of sexual maturation (F1 generation), organ weights, macroscopic pathology, microscopic pathology and sperm evaluations (including motility, count and morphological evaluation). There were no test item related effects on the parameters and end points monitored/evaluated in this study.

In conclusion, once daily oral gavage administration of EXP0700332 to rats at dose levels of up to 1000 mg/kg/day from 10 weeks prior to mating, and then throughout mating, gestation and lactation was well tolerated and had no effect upon the reproductive performance of either the initial (F0) generation or the subsequent (F1) generation. Therefore, under the conditions of this study the no observed effect level (NOEL) was considered to be 1000 mg/kg/day.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 March 2013 to 03 October 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

The Han Wistar rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies. The total number of animals to be used on this study was considered to be the minimum required to properly characterise the effects of the test item. This study was designed such that it does not require an unnecessary number of animals to accomplish its objectives. At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
The oral route of administration was selected for this study as this route has been defined by the Sponsor as a possible route of human exposure. The dose levels were agreed with the Sponsor following evaluation of a previous twenty-eight day repeated dose oral (gavage) toxicity study in the rat (OECD Guideline 407) performed on behalf of the Sponsor.
The dose volume administered to each animal was based on the body weight measurement recorded immediately prior to dosing. The males were dosed daily for 4 weeks, starting 2 weeks prior to mating. The females were dosed daily 2 weeks prior to mating, throughout mating and through to at least Day 4 of lactation.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch No.of test material: E00031-633
- Expiration date of the lot/batch: 30 January 2014
- Purity test date: >/= 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

Species:

rat

Strain:

other: Han Wistar

Details on species / strain selection:

The Han Wistar rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Ltd, Maragate, Kent, UK
- Females (if applicable) nulliparous and non-pregnant
- Age at study initiation: (P) Males: 8-9 weeks; females 7-8 weeks
- Weight at study initiation: (P) Males: 241 - 300 g; Females: 138 - 177 g
- Fasting period before study: no
- Housing: Animals were housed in appropriately sized polycarbonate cages with stainless steel grid tops and solid bottoms. Cages were suspended on moveable racks and fitted with water bottles and integrated stainless steel food hoppers. Prior to pairing for mating, the animals were housed 2 or 3 per sex per cage per group. After mating the animals were re-housed with their original cage mates.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 44-85%
- Air changes (per hr): minimum of 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark cycle
IN-LIFE DATES: From: 02 April 2013 To: 13 May 2013

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Test item dosing formulations were prepared based on a method established at the Test Facility at appropriate concentrations to meet dosage level requirements. The dosing formulations were prepared at appropriate intervals, split into suitable volume aliquots for daily dispensing, stored in a refrigerator set to maintain 4°C and dispensed daily. All formulations were used within the 8 day stability period (as established in Charles River Study No. 430142). The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes prior to and throughout dosing.

VEHICLE

The control formulation, Arachis Oil BP, was prepared at appropriate intervals, stored in a refrigerator set to maintain 4°C and dispensed daily. The prepared control formulation was removed from the refrigerator and stirred for at least 30 minutes prior to dosing and continuously during dosing.

Details on mating procedure:

A few days prior to the initiation of mating, the males will be separated into individual grid bottomed cages. Pairings will be on a one male to one female basis. Animals will be paired in numerical order within groups. Each female will be transferred to the cage of its appropriate co-group male at approximately 1900 hours, where it will remain until mating has occurred or 14 nights have elapsed. Vaginal lavages will be taken daily each morning from the day of pairing until mating has occurred and the stage of oestrus observed in each lavage will be recorded. The day of detection of a copulatory plug in-situ and/or of sperm in the lavage will be designated Day 0 of gestation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate 1 mL samples were collected from the top, middle and bottom (duplicate middle only for control) from each formulation at each sampling time point and were sent to the analytical laboratory. In addition, triplicate 1 mL samples were collected from the top, middle and bottom (triplicate middle only for control) of each formulation at each sampling time point and were retained as backup samples.
At Week 1, additional duplicate and triplicate samples (as described above) from the top and bottom of the control formulation were collected in error. These samples were not analysed.
The results of the sample concentration analyses were considered acceptable if the mean samples concentration results were within ±10% of theoretical concentration. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentration of =10% for each group.

Duration of treatment / exposure:

4 weeks for males and 6-8 weeks for females

Frequency of treatment:

daily

Details on study schedule:

The test and control items were administered once daily by oral gavage at the dose levels 0, 30, 300 and 1000 mg/kg/day. The dose volume administered to each animal was based on the body weight measurement recorded immediately prior to dosing, except during late gestation; from Day 16 of gestation until when parturition was complete, the dose volume was determined by the weight of the animal on Day 16 of gestation.
The first day of dosing was designated as Day 1. The dosing formulations were stirred continuously throughout dosing, commencing at least 30 minutes prior to dosing. The males were dosed daily for 4 weeks, starting 2 weeks prior to mating. The females were dosed daily 2 weeks prior to mating, throughout mating and through to at least Day 4 of lactation.
Females that failed to litter continued to be dosed and underwent terminal procedures at the same time as females that were pregnant and littered. The dose volume used for these animals was based upon the body weight of the animals at their theoretical Day 16 of gestation, up until it was clear that the animal was not pregnant, at which time the dose volume administered was based upon the body weight measurement recorded immediately prior to dosing.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were agreed with the Sponsor following evaluation of a previous twentyeight day repeated dose oral (gavage) toxicity study in the rat (OECD Guideline 407) performed on behalf of the Sponsor.
The test and control items were administered once daily by oral gavage at the dose levels 0, 30, 300 and 1000 mg/kg/day. The dose volume administered to each animal was based on the body weight measurement recorded immediately prior to dosing, except during late gestation; from Day 16 of gestation until when parturition was complete, the dose volume was determined by the weight of the animal on Day 16 of gestation.
The first day of dosing was designated as Day 1. The dosing formulations were stirred continuously throughout dosing, commencing at least
30 minutes prior to dosing. The males were dosed daily for 4 weeks, starting 2 weeks prior to mating. The females were dosed daily 2 weeks prior to mating, throughout mating and through to at least Day 4 of lactation.
Females that failed to litter continued to be dosed and underwent terminal procedures at the same time as females that were pregnant and littered. The dose volume used for these animals was based upon the body weight of the animals at their theoretical Day 16 of gestation, up until it was clear that the animal was not pregnant, at which time the dose volume administered was based upon the body weight measurement recorded immediately prior to dosing.

Positive control:

Not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
All animals were examined for reaction to treatment daily during the course of the study. The onset, intensity and duration of any signs were recorded.

DETAILED CLINICAL OBSERVATIONS:
Once each week starting pretrial, all animals recieved a detailed clinical examination.

BODY WEIGHT:
Body weights were recorded one week prior to the start of treatment. From the start of treatment, the individual body weights were recorded daily.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was measured for both sexes weekly, starting 1 week prior to dosing until pairing for mating.
After mating, the female food consumption was measured over Days 0-7, 7-14 and 14-20 of gestation and Days 0-4 of lactation. Male food consumption did not recommence after pairing for mating.

Oestrous cyclicity (parental animals):

Vaginal lavages were taken daily early each morning from the day of pairing until mating had occured; the stage of of oesrus observed in each lavage was recorded.

Sperm parameters (parental animals):

The epididymides and testes were weighed and underwent histology and histopathogy analysis.

Litter observations:

The females were allowed to litter normally. Any observed difficulty or prolongation of parturition was recorded. The day of birth of the litter (day on which the first pups were born) was designated as Day 0 of lactation. The duration of gestation was calculated.
The numbers of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were counted and examined for the presence of milk in the stomach and for any externally visible abnormalities daily.
Each litter was weighed en masse (by sex) on Days 1 and 4 of lactation.
Where practicable, any pups that were found dead or are killed during lactation were sexed and appropriately examined as above and subject to necropsy procedures and preservation. Pups submitted for necropsy, were examined with a view to diagnosis of the cause of the condition or cause of death. This consisted of external examination, including (where practicable) a check for the presence of milk in the stomach, followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Pups were preserved whole in 10% neutral buffered formalin with organs/tissues in situ.

Postmortem examinations (parental animals):

All adult animals were subjected to a complete necropsy examination which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. The reproductive tracts of all females were examined for signs of implantation, with the number of implantation sites being recorded. In addition the total number of corpora lutea graviditatis in the ovaries of each female was recorded.
Necropsy examinations were conducted by trained technicians and consisted of an external and internal examination and recording of observations for all animals. A veterinary pathologist was available for consultation during normal working hours. The animals were terminated by exposure to carbon dioxide.
The epididymis and testis (males) were the only organs weighed; terminal body weights were used for organ weight analysis. The following tissues were collected and preserved for analysis: epididymis, testis, cervix, pituitary gland, prostate gland, seminal vesicle gland, gross lesions, ovaries, oviducts, uterus and vagina.Histopathological evaluations were performed on the epididymides, ovaries and testes from group 1 (control) and group 4 (high dose level: 1000 mg/kg/day) animals by a veterinary pathologist.

Postmortem examinations (offspring):

Pups submitted for necropsy, were examined with a view to diagnosis of the cause of the condition or cause of death. This consisted of external examination, including (where practicable) a check for the presence of milk in the stomach, followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Pups were preserved whole in 10% neutral buffered formalin with organs/tissues in situ.

Statistics:

Where required to assist with interpretation, tests were applied to determine the statistical significance of observed differences between Control and groups receiving test item. Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the Control group.
Body weight and food consumption (with the exception of females during gestation and lactation) data was analysed for homogeneity of variance using the ‘F-Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test i.e. pairwise comparisons were made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
Organ weight data was analysed as above, and by analysis of covariance (ANCOVA) using terminal body weight as the covariate. In addition, organ weights as a percentage of terminal body weight were also analysed using ANOVA as an exploratory analysis, however following review of the results from these analyses, it was considered unnecessary to include these in the study report.

Reproductive indices:

For each group the gestation index and the fertility index for males and females were evaluated. For each litter and group, the birth index, live birth index and the viability index were evaluated.

Offspring viability indices:

Each litter and group, the birth index, live birth index and viability index were evaluated.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

1 male showed a bald area in group 1 (no test material) and some females showed some changes in fur growth and staining on dorsal neck, however this is was considered not to be related to EXP0700332.
All observations recorded for dams and their pups were common findings for this species and strain, and the distribution of observations across the dose groups indicated no relationship to EXP0700332.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight gains for males and females were similar between the Control animals and the treated animals.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption for males and females were similar between the control and treated animals.

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry 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:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of EXP0700332 by oral gavage.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

The fertility index was 80% or above with 3 females failing to get pregnant:
At 1000 mg/kg/day, 3/10 females passed 1 or more oestruses without mating, versus 0/10 in the 0, 30 and 300 mg/kg/day dose groups. One of these animals (Number 78) passed 3 oestruses in total over the 14 days mating period, and only displayed one equivocal mating sign during this time; this animal did not become pregnant. The other 2 animals (Numbers 74 and 75) mated successfully at the next available oestrus, and as there were no other problems noted with the overall reproductive performance of these animals, or indeed in any of the other animals receiving 1000 mg/kg/day; the distribution of these findings at 1000 mg/kg/day was considered to be incidental.
The fertility index at 1000 mg/kg/day for both males and females was slightly lower than Control (80% versus 90%, respectively), however given the minor magnitude of difference from Control, this findings was considered to be incidental.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

The fertility index at 1000 mg/kg/day for both males and females was slightly lower than Control (80% versus 90%, respectively), however given the minor magnitude of difference from Control, this findings was considered to be incidental.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating performance, fertility indices, corpora lutea and implantation counts and duration of gestation were unaffected by EXP0700332.
At 1000 mg/kg/day, 3/10 females passed 1 or more oestruses without mating, versus 0/10 in the 0, 30 and 300 mg/kg/day dose groups. One of these animals (Number 78) passed 3 oestruses in total over the 14 days mating period, and only displayed one equivocal mating sign during this time; this animal did not become pregnant. The other 2 animals (Numbers 74 and 75) mated successfully at the next available oestrus, and as there were no other problems noted with the overall reproductive performance of these animals, or indeed in any of the other animals receiving 1000 mg/kg/day, the distribution of these findings at 1000 mg/kg/day was considered to be incidental.
The fertility index at 1000 mg/kg/day for both males and females was slightly lower than Control (80% versus 90%, respectively), however given the minor magnitude of difference from Control, this findings was considered to be incidental.

Key result

Dose descriptor:

NOEL

Effect level:

ca. 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive performance

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

All observations recorded for dams and their pups were common findings for this species and strain, and the distribution of observations across the dose groups indicated no relationship to EXP0700332.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

ca. 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

In conclusion, under the conditions of this study the No Observed Effect Level (NOEL) was considered to be 1000 mg/kg/day.

Executive summary:

The purpose of this study was to provide initial information on possible effects on reproduction and/or development from repeated exposure of Han Wistar (Crl: WI (Han)) male and female rats to EXP0700332 beginning before cohabitation, through mating and continuing for at least 28 days (male rats) or through parturition until at least Day 4 of lactation (female rats

Han Wistar rats were randomised into 3 test groups (30, 300 and 1000 mg/kg/day) and one Control group, each containing

10 males and 10 females. The males were treated for 2 weeks prior to mating, then through mating, and until the day prior to necropsy (4 weeks of treatment). Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation (6-8 weeks of treatment).

The following parameters and end points were evaluated in this study: clinical signs, body weights, food consumption, reproductive parameters (mating and pregnancy performance, fertility, maternal care and pup performance), organ weights (testes and epididymides), and gross and microscopic pathological examinations.

Administration of EXP0700332 at dose levels of up to 1000 mg/kg/day was not associated with any changes in clinical observations, body weight gain, food consumption, reproductive function (including maternal care and pup/litter survival), organ weights (males only), or gross or histopathological findings.

In conclusion, under the conditions of this study the No Observed Effect Level (NOEL) was considered to be 1000 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimish 1 - studies were conducted according to the guidelines and to GLP.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

EXP0700332 was conducted to GLP and in accordance to OECD 414. No adverse effects were observed with the NOEL > 1000 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 March 2015 to 10 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch No.of test material: E00031-541
- Expiration date of the lot/batch: 01 November 2016
- Purity test date: > 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature in the dark

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, UK
- Age at study initiation: 9-10 weeks old
- Weight at study initiation: 183 - 264 g
- Housing: 2 animals per cage in appropriately sized suspended polycarbonate/polypropylene cages.
- Diet (e.g. ad libitum): ad libitum throughout study, except during designated procedures
- Water (e.g. ad libitum): ad libitum
- Acclimation period: From arrival until Day 6 gestation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-25 °C
- Humidity (%): 44-57%
- Air changes (per hr): at least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

IN-LIFE DATES: From: 30th March 2015 To: 14th April 2015

Route of administration:

oral: gavage

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations were prepared based on a method established at the Test Facility at appropriate concentrations to meet dosage level requirements. The dosing formulations were prepared at appropriate intervals and were split into suitable volume aliquots for daily dispensing, stored in a refrigerator set to maintain 4°C, and dispensed daily. The dosing formulations were removed from the refrigerator for at least 30 minutes before dosing. The dosing formulations were stirred continuously from at least 30 minutes prior to dosing until dosing was completed each day. Any residual volumes were discarded with the exception of the residues from Day 17 of dosing; these residues were used for formulation analysis sampling; following completion of sampling the residues were discarded.

VEHICLE
Arachis Oil
- Amount of vehicle (if gavage): 4 mL/kg
The control formulation, Arachis Oil BP, was stored in a refrigerator set to maintain 4°C, and dispensed daily. The control formulation was removed from the refrigerator at least 30 minutes prior to dosing. The control formulation was stirred continuously from at least 30 minutes prior to dosing until dosing was completed each day. Any residual volumes were discarded with the exception of the residues from Day 17 of dosing; these residues were used for formulation analysis sampling; following completion of sampling the residues were discarded.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Test item and the control were administered from Days 6-20 of gestation.

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day

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:

24 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were agreed with the Sponsor following evaluation of a previous twenty-eight day repeated dose oral (gavage) toxicity study in the rat (OECD Guideline 407) and a reproduction/ developmental toxicity screening oral gavage study in the rat (OECD Guideline 421; Charles River Study No. 496381) performed on behalf of the Sponsor.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Animals were checked twice a day (once at the start and once at the end of the working day throughout the study) for general health, mortality or moribundity. The animals were not removed from the cage during these observations, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- The animals were removed frm the cage and examined daily from the first day of dosing (Day 6 of gestation)

BODY WEIGHT: Yes
- Body weights were recorded on Day and on Days 6 to 21 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal was quantitatively measured daily from Day 4 to Day 21 of gestation.

WATER CONSUMPTION
- Water consumption was monitored by visual inspection of the water bottles regularly throughout the study.

POST-MORTEM EXAMINATIONS: Yes / No / No data
- Animals were euthanised by carbon dioxide exposure on Day 21 of gestation. All adult animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

Ovaries and uterine content:

The reproductive tract was dissected from the abdominal cavity. The gravid uterus was weighed. The uterus was opened and the contents examined. The fetuses were then removed from the uterus.
The ovaries and uterus were examined for number and distribution of corpora lutea, implantation sites, placentae (size, color or shape) – any abnormalities were recorded as were the numbers of live and dead fetuses and early and late embryonic deaths.

Fetal examinations:

Fetuses were examined for external abnormalities. Late resorptions and dead fetuses were examined for external abnormalities to the extent possible.
Each implant was classified as being a live fetus, a dead fetus (dead full term fetus that showed no sign of maceration), a late embryonic death (macerated tissue identifiable as an embryo or fetus, with recognisable external features such as tail, limbs, mouth and nares present; attached to distinct identifiable placentae), or an early embryonic death (discrete, formless, discolored tissue mass attached to the internal uterine wall; may be of varying size).

Statistics:

Means and standard deviations were calculated for body weight, food consumption and pregnancy data.
Where required to assist interpretations, tests were applied to determine the statistical significance of observed differences between Control and groups receiving test item. All statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group. Body weight and food consumption data were analysed for homogeneity of variance using the ‘F-Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test ie pairwise comparisons were made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal- Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
Following review of the other data obtained on the study, the Study Director considered that no further statistical analysis necessary.

Indices:

Post-implantation loss (%) per litter, group mean fetal weights for live fetuses and group mean litter weight were calculated.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Post dose ploughing in the cage shavings was noted in animals from all dose groups. As this observation was also recorded in control animals it is not considered related to the test item, but may be a result of the oil based vehicle.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

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:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Description (incidence and severity):

The total number of abortions was the same between the control and tested animals.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Pregnancy performance parameters were similar between the control animals and the animals that recieved EXP0700332.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Pregnancy performance parameters were similar between the control animals and the animals that recieved EXP0700332.

Early or late resorptions:

no effects observed

Description (incidence and severity):

Pregnancy performance parameters were similar between the control animals and the animals that recieved EXP0700332.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

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

Description (incidence and severity):

Pregnancy performance parameters were similar between the control animals and the animals that recieved EXP0700332.

Key result

Dose descriptor:

NOEL

Effect level:

ca. 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

histopathology: neoplastic

histopathology: non-neoplastic

maternal abnormalities

mortality

necropsy findings

number of abortions

ophthalmological examination

pre and post implantation loss

total litter losses by resorption

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

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

Description (incidence and severity):

There were no fetal abnormalities and variants, including those indicating the state of skeletal
ossification, that were considered to be related to EXP0700332 administration.

Skeletal malformations:

no effects observed

Description (incidence and severity):

There were no fetal abnormalities and variants, including those indicating the state of skeletal
ossification, that were considered to be related to EXP0700332 administration.

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no fetal abnormalities and variants, including those indicating the state of skeletal
ossification, that were considered to be related to EXP0700332 administration.

Key result

Dose descriptor:

NOEL

Effect level:

ca. 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Administration of EXP0700332 at dose levels of up to 1000 mg/kg/day was not associated with any changes in clinical signs, body weights, food consumption, necropsy findings, pregnancy performance or fetal development. In conclusion, under the conditions of this study the maternal and fetal no observed effect level (NOEL) was considered to be 1000 mg/kg/day.

Executive summary:

The objective of this study was to determine the potential toxicity of EXP0700332 when administered by oral gavage to pregnant rats from implantation until the end of gestation. Dosage levels of 0, 100, 300 and 1000 mg/kg/day were administered to each animal once a day. The following parameters and end points were evaluated in this study: clinical signs, body weights, food consumption, gross necropsy findings, pregnancy performance and fetal examinations. Administration of EXP0700332 at dose levels of up to 1000 mg/kg/day was not associated with any changes in clinical signs, body weights, food consumption, necropsy findings, pregnancy performance or fetal development. In conclusion, under the conditions of this study the maternal and fetal no observed effect level (NOEL) was considered to be 1000 mg/kg/day.

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:

rat

Quality of whole database:

Klimish 1 - conducted to GLP and in accordance with the OECD guideline

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Results from modern GLP studies conducted in accordance with the OECD 414, 416 and 421 guidelines were found to have a NOEL > 1000 mg/kg/day. The test substance (EXP0700332) is not considered to require classification under the terms of CLP Regulation.