Reaction mass of 1,3-diacetoxypropan-2-yl 12-acetoxyoctadecanoate and 2,3-diacetoxypropyl 12-acetoxyoctadecanoate

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2009.01.06 to 2009.10.08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study performed represents a 2-generation reproduction/developmental neurotoxicity combination study design taking into consideration OECD, OPPTS, EU and Japanese testing guidelines. The study was performed in accordance with the study protocol with a number of deviations, none of which adversly affected the ability of the study to detect potential effects of TS-ED 532 and therefore were considered to have had no impact on the scientific integrity of the study. The deviations to study protocol is described in the study report. The study was performed in accordance with GLP regulation.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture Forestry and Fisheries (MAFF), Testing Guidelines for Toxicology Studies (2-1-17), 12 Nohsan No. 8147, November 24, 2000 (partially revised on June 26, 2001).

Deviations:

no

Principles of method if other than guideline:

The study performed represents a combination study design specifically taking into consideration the requirements of the OECD guideline 416 and 426. A combination study design was chosen based on the requirements of using the minimum number of test animals. Further, the combinatory test design was requested by the Danish EPA.

The study was performed to investigate the effects of TS-ED 532 on reproduction, pre-natal development and post-natal development of the rat when administered to two successive generations. Measurement of antiandrogen endpoints (nipple retention at PND 12-15 and anogenital distance at PND 1) was included. The study also investigated the potential of TS-ED 532 to cause developmental neurotoxicity in the offspring. An optionally 3-generation reproduction study was to be performed if effects on reproduction was identified earlier in the study.

The study also incorporated a positive contro group for endocrine disruption (anogenital distance, nipple retention and sexual development) using DEHP (Bis(2-ethylhexyl) phathalate), throughout the F0 generation and undtil sexual maturation of the F1 offspring. The data for DEHP included into this endpoint is limited to anogenital distance, nipple retention and sexual developmen.

Prior to the commencement of this study. the ability of the end points used to assess neurobehavioral effects were validated within the Harlan Laboratories (study described in section 7.9.1 Neurotoxicity - "Developmental neurotoxicity (validation study)".

Additionally, the end points used to assess endocrine disruption (niple retention, anogenital distance and sexual development) were also validated in the Harlan Laboratories (studies described in section 7.8.1 Toxicity to reproduction - "2-generation/developmental neurotoxicity study (preliminary study)" and "Endocrine disruption (validation study").

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

451-530-8

EC Name:

-

Cas Number:

736150-63-3

Molecular formula:

Not applicable

IUPAC Name:

1,3-bis(acetyloxy)propan-2-yl 12-(carboxyoxy)octadecanoate; 2,3-bis(acetyloxy)propyl 12-(acetyloxy)octadecanoate

Details on test material:

- Name of test material (as cited in study report): TS-ED 532
- Lot/batch No.: 4010534806 (item 175540)
- Expiration date of the lot/batch: 2009-12-31

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sprague-Dawley Crl:CD® (SD) IGS BR strain rats from Charles River (UK) Limited, Margate, Kent.
- Age at study initiation: 7 -10 weeks old.
- Weight at study initiation: males weighed 216 to 361g and females weighed 149 to 249 g.
- Fasting period before study: none

- Housing: Initially, all animals were housed in groups of up to four in polypropylene cages with stainless steel grid tops, furnished with softwood flakes (Datesand Ltd., Cheshire, UK). During the mating phase, animals were transferred, on a one male: one female basis within each dose group, to polypropylene grid-floor cages suspended over trays lined with absorbent paper. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually, during gestation and lactation, in polypropylene cages with solid floors and stainless steel lids, furnished with softwood fla kes (Datesand Ltd., Cheshire, UK). For the offspring selected to form the F1 generation, this housing procedure was repeated from when animals were weaned at Day 21 of age. Following weaning, on Day 21 of age, the F0-F1 offspring selected for assessment of developmental neurotoxicity were housed in groups of up to four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. These offspring were divided in to two sets, one set for the majority of behavioural tests and the second set which were used for the testing of naive animals in the water maze on Days 61-62 of age. As far as possible, within each group and each set, animals of the same age were housed together.
Control animals and those animals receiving TS-ED 532 were all housed in the same room; the animals receiving DEHP (positive control) were housed in a separate room to avoid cross-contamination. Following weaning, the F0-F1 offspring selected for the assessment of developmental neurotoxicity were also housed in a separate room to avoid cross-contamination, although they would have had direct exposure to the treated diets prior to weaning.

- Diet (e.g. ad libitum): ground diet ad libitum(Rodent PMI 5002 Diet, BCM IPS Limited, London, UK) (certificate available in the study report)
- Water (e.g. ad libitum): mains drinking water ad libitum (certificate available in the study report)
- Acclimation period: at least 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2ºC. Environmental conditions were continuously monitored by a computerised system and print-outs of hourly mean temperatures and humidities are included in the study records.
- Humidity (%): 55 ±15%. Environmental conditions were continuously monitored by a computerised system and print-outs of hourly mean temperatures and humidities are included in the study records.
- Air changes (per hr): The animals were housed in air-controlled rooms within the Harlan Barrier maintained Rodent Facility with at least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous (07.00 to 19.00 GMT) light and twelve hours darkness.

IN-LIFE DATES: From: 2009.01.06 To:2009.10.08

Administration / exposure

Route of administration:

oral: feed

Vehicle:

arachis oil

Remarks:

The control group received basal diet containing an amount of arachis oil equivalent to the TS-ED 532 added to the high dosage diet, over the same treatment period, to ensure comparable calorific intake.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): The stability and homogeneity of TS-ED 532 in the diet had previously been determined to be stable for a period of at least 23 days. Dietary admixtures were prepared prior to treatment and then prepared in batches ensuring that the availability of last use on the study was within the period of stability for that batch. In general, batches of diets were prepared in advance on a as need basis.

- Mixing appropriate amounts with (Type of food): TS-ED 532 was incorporated into the diet at initial concentrations of 1500, 6000 and 15000 ppm, rising to 20000 ppm and then 25000 ppm as follows. Each concentration of TS-ED 532 was prepared by weighing an appropriate amount of TS-ED 532 out and mixing it with a small amount of the required volume of basal laboratory diet (Rodent PMI 5002 Diet, BCM IPS Limited, London, UK) using a Hobart QE 200 or Robot Coupe mixer. Once this was adequately mixed the formulation was then transferred to a Hobart H800 mixer and mixed with the remaining required volume of diet. The control animals were given laboratory diet treated with amounts of Arachis oil equivalent to the amount of TS-ED 532 added to the high dosage group formulations to ensure comparable calorific intake.
The positive control substance (DEHP) was added in the same manner as detailed for TS-ED 532.

- Storage temperature of food: The diet was stored at ambient temperature in labelled, double plastic bags in labelled, covered plastic bins when not in use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): No vehicle used. Control animals were handled in an identical manner to those receiving TS-ED 532 and were given laboratory diet treated with equal amounts of Arachis oil to TS-ED 532 added to the high dose group to ensure comparable calorific intake

Details on mating procedure:

- M/F ratio per cage: F0 and F1 animals were paired, following subsequent maturation periods, on a 1 male: 1 female basis within each dose group.

- Length of cohabitation: During week 11, following at least ten weeks exposure to the dietary admixtures, all animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.

- Proof of pregnancy: Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating and was Day 0 of pregnancy.

- Further matings after two unsuccessful attempts: No. The protocol allowed for the extension of the mating phase to a third week or the use of a second mate if treatment-related effects were apparent. As mating performance was considered to be good in both generations, these options were not employed in either generation.

- After successful mating each pregnant female was caged (how): Following evidence of mating, or after completion of the pairing period, the males were returned to their original cages and females were transferred to individual cages. Mated females were housed individually during the period of gestation and lactation.

- Pregnacy and parturition: For each generation, pregnant females were observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays.

The following was recorded, where observed, for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition

- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of TS-ED 532 in the dietary admixtures (Rodent PMI 5002 (certified) diet, BCM IPS) was determined by gas chromatography (GC) using an external standard technique.

The concentration of DEHP (positive control) in the dietary admixtures was determined by high performance liquid chromatography (HPLC) using an external standard technique.

The stability and homogeneity of TS-ED 532 in the diet matrix was confirmed for up to 23 days at ambient temperature over the concentration range used by the Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory.

The stability and homogeneity of DEHP in the diet matrix was also confirmed for up to 23 days at ambient temperature at the concentration of used.

Based on the results, it was concluded the schedule for TS-ED 532 and DEHP (positive control) formulations employed on this study was sufficient to give suitable homogeneous and stable formulations for use on the study and that the analytical methods were satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study. The dietary admixtures used on the study were analysed for achieved concentration at selected time points throughout the study by the Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory.

Duration of treatment / exposure:

TS-ED 532 was administered orally by dietary admixture continuously over each generation, including a maturation period of at least 10 weeks, mating, gestation and lactation. Offspring selected for the F1 generation continued to have their respective dietary admixtures between weaning and the start of the formal F1 generation. Control animals were handled in an identical manner to those receiving TS-ED 532 and were given laboratory diet treated with amounts of Arachis oil equivalent to the amount of TS-ED 532 added to the high dose group dietary admixtures to ensure comparable calorific intake. In order to achieve a high dose level that approximated a TS-ED 532 intake equivalent to 1000 mg/kg bw/day, the dietary concentration of TS-ED 532 in diet for this group was reviewed and periodically adjusted during maturation. No further adjustment of dietary level was made during the mating, gestation and lactation phases of the study for practical reasons; animals would be at differing stages of gestation or lactation and have different pregnancy or lactation statuses (i.e. pregnant, non-pregnant, littering, total litter loss) on any particular day of the study making estimation of the correct dietary level difficult and running the risk of some animals receiving less that the target dosage. The TS-ED 532 treated and control F0-F1 offspring selected for post-weaning assessment of developmental neurotoxicity received basal diet from weaning (Day 21 of age) until termination on Day 70 of age.

DEHP (positive control) was administered orally by dietary admixture continuosly to F0 generation rats over a maturation period of at least 10 weeks, and throughout mating, gestation and lactation and subsequently F1 generation rats until the attainment of sexual maturation.

Frequency of treatment:

Continously through the diet.

Details on study schedule:

- F1 parental animals not mated until at least 10 weeks from formal start of F1 generation and approximately 11 weeks after selected from the F1 litters.

- Initial selection of parents from F1 generation occurred at weaning when pups were 21 days of age, however formal commencement of the F1 generation occurred when animals were nominally 28 days of age .

- Age at mating of the mated animals in the study: F0 animals approximately 7-8 weeks at commencement and were paired after 10 weeks of treatment so were approximately 17-18 weeks of age. F1 animals were approximately 4 weeks of age at formal commencement of F1 generation and were therefore approximately 14 weeks of age at mating following the 10 week maturation period.

Doses / concentrations

Remarks:

Doses / Concentrations:
TS-ED 532: 0, 1500, 6000 and 25000 ppm; DEHP (positive control): 10000 ppm.
Basis:
nominal in diet
Mean achieved dosages can be found in Table 3.

No. of animals per sex per dose:

A total of two hundred and eighty animals (one hundre and forty males and one hundred and forty females) were accepted into the study (see Table 1 and 2). The animals were allocated as follows:


Fo animals: 28 male male and 28 female/per dose level (0, 1500, 6000 and 25000 ppm TS-ED 532)
F1 animals: 24 male male and 24 female/dose level, (0, 1500, 6000 and 25000 ppm TS-ED 532)

Fo animals: 28 male male and 28 female (10000 ppm DEHP - positive control)
F1 animals: 24 male male and 24 female (10000 ppm DEHP - positive control)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based upon available data including a preliminary range-finding study (study described in section 7.8.1 - "2-generation reproduction/developmental neurotoxicity study (preliminary study") and recommendation in guidelines.

- Rationale for animal assignment (if not random): A total of twenty-eight male and twenty-eight female F0 animals were allocated to each dose group using a randomisation procedure based on stratified bodyweights and the group mean bodyweights were then determined to ensure similarity between the dose groups. The animals were uniquely identified within the study, by an ear punching system routinely used in these laboratories.
Twenty-four males and twenty-four females were selected from F0 litters from each dose group to form the formal F1 generation. The litters used were chosen to provide a minimal spread of ages within the subsequent F1 generation as possible, while also maintaining as wide a genetic pool within each group as possible. The offspring were selected randomly within each litter, with one male and one female being selected from each litter, where possible. Where insufficient litters were available for offspring selection, additional offspring were selected from the remaining litters.The litters used were chosen to provide a minimal spread of ages within the subsequent F1 generation as possible, while also maintaining as wide a genetic pool within each group as possible; consideration was also given to the need to select sufficient offspring from the remaining offspring for assessment of developmental neurotoxicity.
The offspring selected for pre-weaning assessment of developmental neurotoxicity were selected using random numbers, with either one male and one female being selected for each TS-ED 532 treated and control litter in each generation. As far as possible, the number of male and female offspring was balanced within each group to try to give an even distribution of each sex. Offspring were identified by a within litter tattoo system using the paws and tail and are identified within this report by a combination of their dam number followed by their two digit within litter identifier.
The TS-ED 532 treated and control F0-F1 offspring selected for post-weaning assessment of developmental neurotoxicity were divided into two subsets; one for the majority of testing and the other to provide naive animals for testing in the water maze on Days 61-62 of age. These animals were also selected using random numbers, with, as far as possible, two males and two females being selected from each litter. Due to litter size and unfavourable sex ratios it was not always possible to select the full complement from each litter. Within each group, as far as possible, the number of male and female offspring was balanced within each sub-group and between each sub-group to try to give an even distribution of each sex. Offspring were identified by a within litter tattoo system using the paws and tail in combination with an ear mark corresponding to the dam they were derived from. They are identified within this study by a combination of the dam number followed by their two digits within litter identifier.


- Other: The following overall combined study design was established. TS-ED 53 was administered orally by dietary admixture to three groups each of twenty eight male and twenty eight female F0 Generation rats and subsequently twenty four male and twenty four female F1 Generation rats, over a maturation period of at least 10 weeks, and throughout mating, gestation and lactation for each generation. Animals allocated to low and intermediate dosage groups received intended dietary inclusion levels of 1500 or 6000 ppm respectively. In each generation, animals allocated to the high dosage group initially received an intended dietary inclusion level of 15000 ppm, rising to 20000 ppm and then 25000 ppm during the maturation period and being sustained at the higher inclusion level until termination. This inclusion level is referred to as 25000 ppm throughout the study report. A further group of twenty-eight male and twenty-eight female F0 Generation rats and subsequently twenty-four male and twenty-four female F1 Generation animals received basal diet, containing an amount of Arachis Oil equivalent toTS-ED 532l added to the high dosage diet, over the same treatment period to serve as a control.
For Control and TS-ED 532 treated F0-F1 litters and F1-F2 litters, offspring were selected for pre-weaning assessment of developmental neurotoxicity, with further F0-F1 offspring being subjected to a more extensive post-weaning assessment of developmental neurotoxicity. The offspring selected for the post-weaning assessment of developmental neurotoxicity received basal diet from weaning (Day 21 of age) to termination at Day 70 of age.
As part of the assessment of general and reproductive toxicity over two generations, clinical signs, bodyweight performance, food consumption, oestrous cycles, mating performance, litter data, offspring survival and growth, offspring ano-genital distance and visible nipple counts, sperm assessments, ovarian follicle counts, macroscopic pathological changes at necropsy, selected reproductive organ weights and microscopic histopathological changes of selected organs were monitored. As part of the assessment of developmental neurotoxicity, pre-weaning surface righting, air righting and motor activity and post-weaning motor activity, grip strength, rotor rod performance, assessment of learning and development (water maze performance), startle response and microscopic histopathological changes of the neurological system were monitored for F0 - F1 offspring. Further measurements of pre-weaning motor activity was added to the study and assessed in F1 - F2 offspring.


- Other: Due to the necessity of performing offspring behavioural observations with the operator being unaware of the treatment level, the study was performed “blind” for control animals and animals receiving TS-ED 532. Increasing group number therefore does not correspond to increasing dosage and, for ease of interpretation, data are presented in ascending dosage order for these groups in the tables and appendices rather than group order. The identity of the positive control group (DEHP) was known, as it was housed in a different room to avoid cross-contamination, but as these animals were required as a positive control for endocrine disruption and not developmental neurotoxicity, this did not affect the “blind” performance of the behavioural tests.

Positive control:

A positive control group (DEHP) for the end points used to assess endocrine disruption (nipple retention and anogenital distance) was included in the study.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Morbidity and mortality observations for adult animals and behavioural offspring were performed twice daily during the week and once daily on weekends and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All adult animals were examined for overt signs of toxicity, ill-health or behavioural change once daily. A more detailed check of clinical condition was made on a weekly basis. Clinical signs for all animals were normally recorded on weekly basis but once an observation was observed, more frequent recording of clinical signs was generally instigated for individual animals until the clinical sign was no longer present. For the offspring selected for post-weaning assessment of developmental neurotoxicity, clinical signs were generally recorded on a daily basis.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were initially recorded for adult animals in Week 1 (Day 1, prior to formal treatment) for each generation and then weekly throughout the maturation phase of each generation and continuing for males until termination. Following pairing, F0 and F1 females were weighed daily until mating was evident. Bodyweights for females showing evidence of mating were recorded on Days 0, 7, 14 and 21 post coitum and for females that littered on Days 1, 4, 7, 14 and 21 post partum.
For the F0-F1 offspring selected for post-weaning assessment of developmental neurotoxicity, bodyweights were recorded on Days 28, 35, 42, 49, 56, 63 and 70 days of age. Additional bodyweights were also recorded for both F1 animals and F0-F1 offspring selected for post-weaning assessment of developmental neurotoxicity, when sexual maturation was attained. Bodyweight performance of the offspring selected to form the F1 generation was also monitored between weaning and the formal commencement of the F1 generation. Bodyweight of the F0-F1 offspring selected for post-weaning assessment of developmental neurotoxicity was recorded when many of the neurological behavioural assessments were performed; i.e. air righting, rotor rod, grip strength, motor activity and startle reflex.
Bodyweight was generally recorded for all adult animals and offspring in both generations and for F0-F1 offspring selected for post-weaning assessment of developmental neurotoxicity on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, for both generations, weekly food consumption was recorded for each cage of adults during the maturation period and was re-instigated for males once they had returned to their home cage following mating/pairing. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0 - 7, 7 - 14 and 14 - 21 post coitum. For females that littered, food consumption was recorded for the period covering Days 1 - 4, 4 - 7, 7 - 14, 14 - 21 post partum. It should be noted that food consumption during the last week of lactation will increasingly be influenced by the growing offspring directly eating the diet and therefore can not be considered to represent food consumption of the lactating female per se during this period.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, values for achieved intake based on nominal TS-ED 532 concentration (ppm) and DEHP (positive control) concentration were calculated using group values for food intake and bodyweights.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Water intake was observed daily by visual inspection of water bottles for any overt change. No significant differences between groups were observed that were considered to necessitate the formal gravimetric recording of water intake.

Oestrous cyclicity (parental animals):

Estrous cyclicity: Prior to pairing of females for the F0 and F1 mating phases, a vaginal smear was taken daily for twenty-one days and a sample was placed on a glass slide.Thesmears were allowed to dry and then stained using a diluted giemsa stain. The smears were examined microscopically and the stage of oestrous was recorded. Where possible, oestrous cycles were classified according to the following criteria:
Normal oestrous - The pattern of daily stages of oestrous show a four to five day cycle, which is generally repeated over 21 days.
Extended oestrous - The observation of a predominance of epithelial/cornified cells for more than two days for more than one oestrous cycle.
Extended dioestrous - The predominant cell type is the leucocyte for more than three consecutive days over more than one oestrous cycle.
Irregular cycle - An irregular length of oestrous cycle is observed over the 21 day evaluation period.
Acyclic - No evidence of an oestrous cycle is observed over the 21 day evaluation period.

Evaluation of Oocyte Number: Slides of the ovaries from ten control and ten 25000 ppm TS-ED 532 treated females from the F1 generation were prepared by serial sectioning of the tissue and selection of every tenth section for mounting on a glass slide. Each slide was stained with haematoxylin and eosin. The sections of ovary were then examined microscopically and visible oocytes were identified and classified according to the following criteria:
Small follicles (S) - Range from a single oocyte to an oocyte with an associated layer of granulosa cells.
Medium follicles (G) - Range from oocytes with more than a single layer of granulosa cells to oocytes with multiple layers of granulosa cells.
Large follicles (A) - Range from oocytes with micro cavitation representing the early follicular antrum to Graafian follicles.

Sperm parameters (parental animals):

At necropsy of adult F0 and F1 males the following evaluations were performed:
i) The left testis and epididymis were removed, dissected from connective tissue and weighed separately.
ii) For the testis, the tunica albuginea was removed and the testicular tissue stored frozen at approximately -20ºC. At an appropriate later date the tissues were thawed, re-weighed and homogenised in a suitable saline/detergent mixture. Samples of the homogenate were stained with a DNA specific fluorescent stain and a sub-sample was analysed for numbers of homogenisation resistant spermatids.
iii) For the epididymis the distal region was incised and a sample of the luminal fluid collected and transferred to a buffer solution for analysis of sperm motility and sperm morphology. Approximately 200 individual sperm were assessed using an automated semen analyser, to determine the number of motile, progressively motile and non-motile sperm.The characteristics of motile sperm were also identified using the computer assisted sperm analyser (Hamilton-Thorne TOX IVOS system).
iv) A sample of semen was preserved in formalin and then stained with eosin. A sub-sample was placed on a glass slide with a coverslip and a morphometric analysis of 200 sperm was performed manually.
v) The cauda epididymis was separated from the body of the epididymis, and then weighed. The cauda epididymis was then frozen at approximately -20ºC. At an appropriate later date the tissues were thawed, re-weighed and homogenised in an appropriate saline/detergent mixture. Samples of the homogenate were stained with eosin and a sub-sample was analysed for homogenisation resistant spermatids.

For the F0 and F1 generation, sperm and spermatid assessments were restricted to control males and males receiving 25000 ppm TS-ED 532.
For the F0 generation, sperm and spermatid assessments were restricted to control males and males receiving 10000 ppm DEHP (positive control).

The following characteristics were recorded for individual sperm analysis:
i) Concentration (millions/ml)
ii) Percentage motility
iii) Sperm morphology
iv) Homegenisation resistant spermatid counts

Litter observations:

STANDARDISATION OF LITTERS
- No standardisation of litter size performed was performed.

PARAMETERS EXAMINED
For each generation, on completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. As far as possible all dead offspring were removed from the litter and subjected to a necropsy examination. All surviving offspring within each litter were individually identified by tattoo (using paws and tail) on Day 1 post partum. For each litter the following was recorded:
i) Number of offspring born.
ii) Number of offspring alive recorded daily and reported on Day 1, 4, 7, 14, 21 post partum. On Days 1, 4, 7, 14 and 21, the sex of individual offspring was recorded.
iii) Clinical condition of offspring from birth to weaning.
iv) Individual offspring bodyweight on Day 1, 4, 7, 14 and 21 post partum.
v) Necropsy findings of offspring.
vi) Physical development. In each generation, ano-genital distance was measured for all offspring on Day 1 of age and numbers of visible nipples were counted for all offspring on Days 11 to 15 of age. The protocol allowed for other landmarks of development (i.e. eye opening, tooth eruption) to be measured in F1-F2 offspring if effects on offspring bodyweight for the F0-F1 offspring were suspected. No obvious adverse effect on bodyweights was apparent during littering for F0-F1 offspring receiving TS-ED 532 and therefore the addition of further landmarks for offspring development was not considered necessary. further, ) surface rigthing reflex was determined on Day 1 and 2.


GROSS EXAMINATION OF DEAD PUPS
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Following completion of the female lactation phase in each generation, the F0 and F1 male animals were killed and examined macroscopically
- Maternal animals: Surviving adult F0 and F1 females and their unselected offspring were killed and examined macroscopically on Day 21 of lactation/age.

GROSS NECROPSY
- Where possible, all adult F0 and F1 animals and the unselected offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. Organ weights for the adult animals and offspring were recorded as required. In addition, the corpora lutea of all ovaries from pregnant females were counted at necropsy. The uterine implantation sites were also counted and, in the case of non-pregnant females, the procedure was enhanced by staining the uteri with a 0.5% ammonium polysulphide solution where applicable.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were removed from all control and TS-ED 532 treated adult animals at the end of each generation. Organs were dissected free from fat and weighed before fixation, with the exception of the thyroid which was weighed post-fixation:
Adrenals, Brain, Left cauda epididymis, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary, Prostate, Seminal vesicles (with coagulating gland and fluids), Spleen, Testes, Thymus, Thyroid gland (including parathyroids), Uterus (with cervix and oviducts).

The following tissues were preserved from all F0 and F1 males and females from each dose group, in buffered 10% formalin:
Adrenals, Prostate, Coagulating gland, Right Epididymis (preserved in Bouins fluid and then in 70% IMS after forty-eight hours), Ovaries, Right Testis (preserved in Bouins fluid and then in 70% IMS after forty-eight hours), Pituitary, Seminal vesicles, Uterus (with Oviducts) and Cervix, Vagina, Gross lesions.

All tissues were despatched to TUPI Manufacturing, Diss, England (the test site responsible for histology processing) for processing (Principal Investigator: Ross Trindall). The tissues from all control and 25000 ppm TS-ED 532 treated F0 and F1 adult animals, any adult animal dying during the study and any 1500 ppm and 6000 ppm TS-ED 532 treated adult animals which failed to mate or that did not achieve a pregnancy were processed. Tissues were prepared as paraffin blocks, sectioned at a nominal thickness of 5μm and stained with haematoxylin and eosin for subsequent microscopic examination. In addition, sections of the testes and epididymides from all control and 25000 ppm TS-ED 532 treated F0 and F1 adult males were also stained with Periodic Acid-Schiff (PAS) stain and examined. The full pathology phase report is included in the test report.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at weaning (Day 21 days of age).The F0-F1 offspring selected for the post-weaning assessment of developmental neurotoxicity were killed at Day 70 of age. With the exception of those offspring selected for whole body perfusion, the offspring were terminated via carbon dioxide asphyxiation and then subjected to a macroscopic necropsy. Brain weights were recorded as required. The F0-F1 behavioural offspring selected for whole body perfusion were killed by intravenous overdose of sodium pentobarbitone followed immediately by perfusion with glutaraldehyde/paraformaldehyde fixative via the heart for in-situ preservation of neural tissues. Macroscopic examination of the animals was more limited in comparison to their non-perfused counterparts due to the nature of the perfusion technique. As far as possible, the offspring selected for perfusion was the one which had been used for the majority of behavioural testing with a representative coming from most litters.

- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: The unselected offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.


GROSS NECROPSY
- Where possible, the unselected offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. Organ weights for offspring were recorded as required.

HISTOPATHOLOGY / ORGAN WEIGTHS
Where possible, one male and one female offspring from each F0-F1 and F1-F2 litter, had the following organs removed and weighed:
Brain, Spleen, Thymus, Uterus.

In addition, samples of the following tissues from all perfused animals at Day 70 of age, and additionally the brains of selected offspring at Day 21, were preserved in fixative for histopathology investigations. All tissues from the perfused animals were despatched to TUPI Manufacturing, Diss, England and where appropriate, routinely processed to paraffin wax/resin and stained with haematoxylin and eosin and examined microscopically. Additionally further slides were prepared for the brain and stained with Cresyl Violet/Luxol Fast blue. Histopathological processing was restricted to control and TS-ED 532 (25000 ppm) perfused animals, with the exception of the brain, which was processed at least to block stage for all perfused animals to prevent shrinkage of the neurological tissues. For the perfused offspring the post-fixation brain weight was also recorded by the processing laboratory.

- Brain at levels (8 sections) to include olfactory bulb, forebrain, centre of cerebrum (including hippocampus), midbrain, cerebellum, pons and medulla oblongata.
- Dorsal root ganglia (cervical and lumbar sections).
- Dorsal and ventral root fibres (longitudinal cervical and lumbar sections).
- Eyes (longitudinal sections).
- Optic nerve (longitudinal sections).
- Peroneal nerve (longitudinal and transverse sections).
- Sciatic nerve - proximal (longitudinal and transverse sections).
-Sural nerve - (longitudinal and transverse sections).
- Tibial nerve - proximal (at the knee) and calf muscle branches (longitudinal and transverse sections).
- Skeletal (calf) muscle (transverse and longitudinal sections).
- Spinal cord (longitudinal and transverse thoracic cervical and lumbar sections).

All prepared slides were despatched to PMM Associates, Edinburgh, Scotland (the Test site for histopathology) for examination by the Study Pathologist, (and Principal Investigator) Peter Millar. For non-perfused animals, microscopic examination was restricted to all control and 25000 ppm TS-ED 532 treated adult animals, adult animal dying during the study and 1500 ppm and 6000 ppm TS-ED 532 treated adult animals which failed to mate or that did not achieve a pregnancy. For perfused animals, microscopic examination was restricted to all control and offspring at 25000 ppm TS-ED 532 Morphometric measurements of selected regions of the brain were performed by the Study Pathologist. The specific regions measured were at the discretion of the Study Pathologist based on experience and the specific findings observed; these are detailed in the raw data and Principal Investigator Report. These measurements did include representative locations within the neocortex, hippocampus and cerebellum. The full pathology phase report is included in the test report.

Statistics:

Statistical analyses were performed on the following data with the results of these analyses being presented on the appropriate tables:
Bodyweight, bodyweight change, food consumption, litter data, implantation losses and survival indices, litter weights, sex ratio (percentage males), ano-genital distance, nipple counts, sexual maturation (vaginal opening, balano preputial separation), motor activity, grip strength, rotor rod, water maze, auditory startle, absolute and bodyweight relative organ/brain weights, sperm concentration and motility, homogenisation resistant spermatid counts, sperm morphology and oocyte assessments (follicle counts).

Data were initially assessed for homogeneity of variance using Levene’s test. Where variances were shown to be homogenous, a parametric assessment of the data was performed using one way analysis of variance (ANOVA), which if significant was followed by pairwise comparisons using Dunnett’s test. Where Levene’s test showed unequal variances, the data were analysed using non-parametric methodology: Kruskal-Wallis ANOVA which, if significant, was followed by Mann-Whitney ‘U’ test.

Probability values (p) are presented as follows:
p ≤ 0.001 ***
p ≤ 0.01 **
p ≤ 0.05 *
p > 0.05 (not significant)

Reproductive indices:

Oestrous cycle: The stage of oestrus for adult females according to critera described in test report.

Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

Fertility indices: Calculated as mating index (%) and pregnancy index(%).

Gestation length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition. Where the start of parturition occurred overnight, the total was adjusted by adding half a day.

Parturition index (%).

Offspring viability indices:

Implantation losses: Calculated as % pre-implantation loss and % post-implantation loss.

Live birth and viability indices: Calculated as live birth index(%), viability index 1,2,3,4,5 (%).

Sex ratio (% males).

Physical Development of the Offspring: For ano-genital distance on Day 1 of age and visible nipple counts between Days 11 and 15, the litter was regarded as the standard unit of assessment. Individual mean litter values are presented for the ano-genital distance and nipple counts for each sex. Group mean values and standard deviations were calculated from these litter means using all litters successfully reared to weaning.

Sexual Maturation: The individual day and bodyweight at attainment of sexual maturity (vaginal opening for females, balano-preputial separation for males) for all offspring selected to form the F1 generation and also for F0-F1 offspring selected for post weaning assessment of developmental neurotoxicity. Group mean values are calculated separately for the F1 generation and F0-F1 offspring using data from all animals.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No adverse effect of treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No adverse effect of treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No adverse effect of treatment.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No adverse effect of treatment.

Other effects:

no effects observed

Description (incidence and severity):

Test substance intake: No adverse effect of treatment.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No adverse effect of treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No adverse effect of treatment.

Reproductive performance:

no effects observed

Description (incidence and severity):

No adverse effect of treatment.

Details on results (P0)

Mortality:
There were no deaths amongst adult animals receiving TS-ED 532 at 1500, 6000 or 25000 ppm, in either generation, which were considered to be related to treatment.
One F0 female receiving TS-ED 532 at 25000 ppm was killed for animal welfare considerations due to a decline in clinical condition. Clinical signs included dehydration, lethargy, emaciation, hunched posture, tip-toed gait and staining around mouth. Macroscopic necropsy examination indicated that the animal had sustained a suspected broken jaw and, while the aetiology of this physical trauma was unknown, this death was considered to be incidental and unrelated to treatment.
One F0 control female and one F0 female receiving TS-ED 532 at 6000 ppm were killed due to adverse clinical condition around the time of expected parturition. Both deaths were considered to be related to difficulties with the birth of their litters and, in isolation, were considered to be unrelated to treatment.

Clinical Observations:
Neither the type, incidence or distribution of clinical signs observed for adult animals indicated any adverse effect of TS-ED 532 at 1500, 6000 or 25000 ppm.
As previously discussed, one F0 female receiving TS-ED 532 at 25000 ppm showed dehydration, lethargy, emaciation, hunched posture, tip-toed gait and staining around mouth prior to being killed for animal welfare considerations. Necropsy examination indicated that the animal had sustained a suspected broken jaw and the decline in clinical condition was considered to be unrelated to treatment.
One control F0 female showed hunched posture, pilo-erection, tip-toed gait, lethargy, pallor of the extremities and vaginal discharge on Day 97 of the F0 generation. Another female, receiving TS-ED 532 at 6000 ppm, showed hunched posture, pilo-erection, staining around the snout, staining around ano-genital region and pallor of the extremities on Day 99 of the F0 generation. As previously discussed, these clinical signs coincided with the time of expected parturition and were considered to reflect difficulties with the birth of their respective litters rather than an effect of treatment.

Bodyweight:
No adverse effect of treatment on mean bodyweight or bodyweight change was apparent in either generation for males receiving TS-ED 532 at 1500,6000 or 25000 ppm.
No adverse effect of treatment on mean bodyweight or bodyweight change prior to pairing, during gestation or during lactation was apparent in either generation for females receiving TS-ED 532 at 1500, 6000 or 25000 ppm.

Food Consumption:
No adverse effect of treatment on mean food consumption was apparent in either generation for males receiving TS-ED 532 at 1500, 6000 or 25000ppm.
No adverse effect of treatment on mean food consumption was apparent in either generation for females receiving TS-ED 532 at 1500, 6000 or 25000 ppm during the maturation, gestation or lactation phases of the study.
Food consumption of F0 females receiving TS-ED 532 at 25000 ppm was significantly lower than the concurrent control during the last week of lactation. Food consumption during this last week is heavily influenced by the litter, as the maturing offspring increase demand on the dam for milk production and also consume the diet directly. In the absence of any previous effect on food intake on the F0 females and any similar effect on the F1 females, this difference was considered to be incidental and unrelated to treatment.
Higher food intake, compared to control, was observed for F0 females receiving TS-ED 532 at 6000 ppm during the last week of gestation and the last week of lactation; differences attaining statistical significance. Similar statistically significant increases in food intake were observed for F1 females at this inclusion level during the last two weeks of gestation and Days 4 to 7 of lactation. For F1 females receiving TS-ED 532 at 1500 ppm, statistically significant higher food intake was also observed for the last week of gestation and the first week of lactation. Food consumption at both of these inclusion levels had a tendency to be marginally higher than control throughout the study but, overall, in the absence of any consistent dosage relationship and the absence of any similar increases at the highest inclusion level, these differences were considered to reflect normal biological variation and were unrelated to treatment with TS-ED 532.

Food conversion efficiency:
No adverse effect of treatment on the efficiency of food utilisation during the pre-pairing or post pairing phases of the study was apparent in either generation for males receiving TS ED 532 at 1500, 6000 or 25000 ppm.
No adverse effect of treatment on the efficiency of food utilisation during the pre-pairing phase of the study or during the first two weeks of gestation was apparent in either generation for females receiving TS-ED 532 at 1500, 6000 or 25000 ppm.

Test substance intake:
See Table 3 below.

Oestrous Cycle Assessment:
There were no obvious adverse effects of treatment on the oestrous cycles of females receiving TS-ED 532 at 1500, 6000 or 25000 ppm in either generation.

Oocyte Assessments:
The mean number of small, medium or large oocyte follicles in the ovaries for F1 females receiving TS-ED 532 at 25000 ppm did not indicate any adverse effect of treatment when compared with concurrent control.

Sperm Analysis:
No adverse effect of treatment on sperm concentration, motility or morphology was apparent for males receiving TS-ED 532 at 1500, 6000 or 25000 ppm in either generation.
For F0 males receiving TS-ED 532 at 25000 ppm, homogenisation resistant spermatid counts for the epididymides and testes were statistically significantly lower than concurrent control. However there was no similar decrease for F1 males, where exposure of the male reproductive system to TS-ED 532 occurred at a much earlier stage of development. There were also no effects on the remaining sperm parameters in either generation, number of implantations and litter size in either generation or evidence of histopathological change for reproductive tissues in either generation. The decrease in F0 males homogenisation resistant spermatid counts was considered to be incidental and of no toxicological significance and in view of this an investigation of spermatid counts at lower exposure levels was considered unnecessary.

Mating Performance, Fertility and Gestation Length:
There were no obvious adverse effects of treatment on mating performance, fertility or gestation length of females receiving TS-ED 532 at 1500, 6000 or 25000 ppm in either generation. While the number of F0 females with live offspring was marginally lower than anticipated at 25000 ppm TS-ED532, there was no similar occurrence in the following F1 generation and this finding was therefore considered coincidental and unrelated to treatment.

Adult Organ Weights:
Assessment of intergroup differences in absolute and bodyweight-relative organ weights for males receiving TS-ED 532 at 1500, 6000 or 25000 ppm did not reveal any obvious adverse effects of treatment in either generation. For F0 males receiving TS-ED 532 at 6000 ppm, absolute and bodyweight-relative weight for the left testis was statistically significantly higher than control. In the absence of any significant increase for the right testis for these males or both testes for F1 males, compared to control, or any treatment related histopathological change, the higher F0 testis weight was considered to be incidental and unrelated to treatment.
Assessment of intergroup differences in absolute and bodyweight-relative organ weights for females receiving TS-ED 532 at 1500, 6000 or 25000 ppm did not reveal any consistent effect of treatment over the two generations.
For F0 females receiving TS-ED 532 at 25000 ppm, absolute and bodyweight-relative thyroid weights were significantly higher than concurrent control, with differences attaining statistical significance. There were, however, no statistically significant differences in thyroid weights for F1 females or for males in either generation at this inclusion level. The F1 females would have had direct consumption to TS-ED 532 in the diet at a much younger age than their female F0 counterparts, as they would have been directly consuming the diet by the third week of age (while still with the lactating parent female) and due to their low bodyweight at this stage of the study were probably receiving a relatively high intake of TS-ED 532 in terms of mg/kg bw/day. Given the earlier and potentially higher exposure of the F1 females to TS-ED 532 and the absence of any accompanying effect on thyroid weights for these animals, the increase in thyroid weights for F0 females appears fortuitous and this finding was therefore considered to be incidental and of no toxicological significance.
Additionally, for F0 females receiving TS-ED 532 at 25000 ppm or 6000 ppm, absolute uterine weight was significantly lower than control; values when adjusted for the bodyweight were not significantly different from control and this finding was therefore considered to be coincidental and to reflect normal biological variation. At 1500 ppm TS-ED 532 bodyweight-relative brain weight was statistically significantly lower than control. In the absence of any dosage relationship, and any effect on absolute brain weight (a more accurate indicator of effect for this organ), this was considered to be incidental and unrelated to treatment.

Gross pathology:
Neither the type, incidence or distribution of necropsy observations in adult males in either generation indicated any adverse effect of treatment with TS-ED 532 at dietary concentrations of 1500, 6000 or 25000 ppm.
At 25000 ppm TS-ED 532, the brains of three F0 males were observed to be surrounded by clear fluid at necropsy, the brain from one of these animals was also misshapen. One control animal was also observed to have fluid surrounding the brain. In the absence of any evidence of treatment-related histopathological change or any similar findings for F0 females or for either sex in the F1 generation (where the animals are exposed at a much earlier stage of brain development), this finding was considered to be incidental and unrelated to treatment.
Neither the type, incidence or distribution of necropsy observations in adult females in either generation indicated any adverse effect of treatment with TS-ED 532 at dietary concentrations of 1500, 6000 or 25000 ppm. The majority of macroscopic necropsy findings were limited to decedent animals; these deaths, as previously discussed, were considered to be unrelated to treatment.

Histopathology:
There were no histopathological findings observed for adult males or females that were considered to indicate an adverse effect of exposure to TS-ED 532 at a dietary inclusion level of 25000 ppm in either generation.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

>= 1 159 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No effect of treatment up to highest dietary concentration (25000 ppm) on general toxicity parameters and reproductive parameters. The mean achieved dosage was 1159 mg/kg bw/day.

2

Dose descriptor:

NOAEL

Effect level:

>= 1 392 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effect of treatment up to highest dietary concentration (25000 ppm) on general toxicity parameters and reproductive parameter. The lowest mean achieved dosage was 1392 mg/kg bw/day (maturation period).

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No adverse effect of treatment.

Mortality / viability:

no mortality observed

Description (incidence and severity):

No adverse effect of treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No adverse effect of treatment.

Sexual maturation:

no effects observed

Description (incidence and severity):

No adverse effect of treatment.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No adverse effect of treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No adverse effect of treatment.

Histopathological findings:

no effects observed

Description (incidence and severity):

No adverse effect of treatment.

Details on results (F1)

Litter responses:
There were 27, 25, 27 and 24 pregnant F0 females at 0 (Control), 1500, 6000 and 25000 ppm TS-ED 532 respectively. One control F0 female and one F0 female receiving TS-ED 532 at 6000 ppm were killed due to suspected problems with parturition and one F0 female receiving TS-ED 532 at 25000 ppm showed total litter loss post partum. In total, there were 26, 25, 26 and 23 F0 females at 0 (Control), 1500, 6000 and 25000 ppm TS-ED 532 respectively that successfully reared young to weaning.
There were 22, 24, 23 and 24 pregnant F1 females at 0 (Control), 1500, 6000 and 25000 ppm TS-ED 532 respectively. One control F1 female and one F1 female receiving TS-ED 532 at 6000 ppm showed total litter loss post partum. In total, there were 21, 24, 22 and 24 F1 females at 0 (Control), 1500, 6000 and 25000 ppm TS-ED 532 respectively that successfully reared young to weaning.

Litter size, sex ratio and viability:
There was no adverse effect of treatment on corpora lutea count, numbers of implantation sites or litter size at birth for females receiving TS-ED 532 at 1500, 6000 or 25000 ppm in either generation. Subsequent survival of the offspring to weaning was similarly unaffected at these dietary concentrations by either maternal treatment or by more direct treatment when the offspring consumed the diet as they approached weaning in both generations. Sex ratio (assessed as percentage males) at birth and subsequently to Day 21 of age was similar to concurrent control in all TS-ED 532 treatment groups over both generations, indicating no selective effect of treatment on survival for either sex.
As previously mentioned, one control F1 female, one F1 female receiving TS-ED 532 at 6000 ppm and one F0 female receiving TS-ED 532 at 25000 ppm showed total litter loss post partum. As there was no consistency between the two generations and also no increase in mortality for offspring for those litters surviving to weaning in either generation, these isolated occurrences were considered to be incidental and unrelated to maternal treatment.
Higher post-implantation loss was observed for F0 females receiving TS-ED 532 at 25000 ppm, compared to control, with differences attaining statistical significance. To a certain extent, the differences observed may reflect particularly good performance for the control F0 females; post-implantation loss was about half of that observed for control females from the F1 generation. There was no subsequent significant difference in litter size at birth for the F0 females at 25000 ppm and no similar increase in post-implantation loss for F1 females at this dosage. In view of this the higher implantation loss observed for the 25000 ppm TS-ED 532 F0 females was considered to be incidental and unrelated to treatment.

Clinical signs:
At 1500, 6000 or 25000 ppm TS-ED 532, the type, incidence and distribution of clinical observations observed for offspring in both generations were consistent with that normally expected in offspring of the age examined and did not indicate any adverse effect of treatment.

Body weight:
There was no adverse effect of treatment with TS-ED 532 on litter weights, offspring bodyweight on Day 1 for either sex or subsequent bodyweight gain of each sex to weaning (Day 21 of age) at dietary concentrations of 1500, 6000 or 25000 ppm in either generation.
For F0-F1 offspring at 25000 ppm TS-ED 532 litter weights were slightly lower than control between Days 14 and 21 with differences attaining statistical significance. In the absence of any statistically significant effect on F0-F1 offspring bodyweights or bodyweight gain, these differences were considered to principally reflect the slight difference in litter size compared to control and were, therefore, considered to be of no toxicological significance.
For F1-F2 offspring at 1500 and 25000 ppm TS-ED 532 bodyweight gain was slightly lower than control for both sexes between Days 7 and 14; differences attaining statistical significance. There was, however, no statistically significant difference in bodyweight at this age for either sex, nor were there any statistically significant differences from control in subsequent bodyweight gain or bodyweight for either sex to weaning. These differences may reflect normal biological variation and were not considered to indicate an adverse effect of treatment.

Sexual maturation:
There were no obvious adverse effects of treatment on the sexual maturation of either sex for F1 animals receiving TS-ED 532 at 1500, 6000 or 25000 ppm. There were also no obvious adverse effects of treatment on the sexual maturation of either sex for the untreated F1 offspring, retained for behavioural assessments, which were derived from females that received TS-ED 532 at 1500, 6000 or 25000 ppm.
For the positive control (DEHP), the attainment of vaginal opening for F1 females and balano-preputial separation for F1 males was clearly delayed for animals receiving DEHP at 10000 ppm, in comparison to control. Bodyweight at the time of attainment for females was greater than control, reflecting the delay in attainment, with differences attaining statistical significance.

Offspring growtn and development:
There was no adverse effect of treatment with TS-ED 532 on mean ano-genital distance on Day 1 of age for either sex at dietary concentrations of 1500, 6000 or 25000 ppm.
For F0-F1 males at 1500 ppm, mean ano-genital distance on Day 1 of age was slightly shorter than concurrent control; difference attaining statistical significance. In the absence of any similar decrease at 25000 ppm TS-ED 532, this decrease weas considered to reflect normal biological variation and were considered to be unrelated to treatment.
For F1-F2 females, mean ano-genital distance on Day 1 of age was slightly shorter than concurrent control; difference attaining statistical significance. In the absence of any similar decrease at 25000 ppm TS-ED 532, this decrease weas considered to reflect normal biological variation and were considered to be unrelated to treatment
There was no adverse effect of treatment with TS-ED 532 on visible nipple counts on Days 11 to 15 of age for either sex at dietary concentrations of 1500, 6000 or 25000 ppm in either generation.
There was no adverse effect of treatment with TS-ED 532 on visible nipple counts on Days 11 to 15 of age for either sex at dietary concentrations of 1500, 6000 or 25000 ppm in either generation. For F0-F1 female offspring at 25000 ppm TS-ED 532 visible nipple counts were marginally higher than control at Day 11 of age, although differences did attain statistical significance. No significant increase from control was observed for these offspring for the subsequent days of assessment (Days 12 to 15), neither was there any similar increase from control observed for the F1-F2 offspring. The higher visible nipple count observed for the F0-F1 female offspring on Day 11 was therefore considered to be incidental and unrelated to treatment.
For the positive control group (DEHP), male F0-F1 offspring at 10000 ppm DEHP, mean ano-genital distance on Day 1 of age was shorter than control; differences attaining statistical significance. For the positive control group (DEHP), male F0-F1 offspring at 10000 ppm DEHP, visible nipple counts were higher than control at Day 15 of age, with differences attaining statistical significance. While no significant differences were observed previous to this, mean values were marginally higher than control. The F0-F1 offspring at this dosage were noticeable smaller than their control counterparts and this failure to detect significant difference in visible nipple count prior to Day 15 of age may reflect immaturity of the male offspring.

Organ weight:
Assessment of intergroup differences in absolute and bodyweight-relative brain, spleen and thymus weights for male offspring receiving TS-ED 532at 1500, 6000 or 25000 ppm did not reveal any obvious adverse effects of treatment in either generation.
For female offspring receiving TS-ED 532 at 25000 ppm, absolute and bodyweight-relative spleen weights were statistically lower than control in both generations (see Table 4 and 5) . Statistically lower absolute and bodyweight-relative spleen weights were observed for F1-F2 females at 1500 ppm TS-ED 532. In the absence of any effect on spleen weight or any treatment related histopathological change for adult F1 females, the lower offspring spleen weight observed was considered to be of no long term toxicological significance. At 6000 ppm TS-ED 532 bodyweight-relative uterus weights of F0-F1 offspring were statistically significantly lower than control although absolute weights were not similarly affected. In the absence of any effects for F1-F2 offspring at this dosage, or any similar effect at 25000 ppm TS-ED 532, this was considered to be incidental and unrelated to treatment.

Gross pathology:
The macroscopic abnormalities observed for both decedent and terminal kill offspring (F0-F1 offspring and F1-F2 offspring) were typical for the age examined and neither the incidence or distribution of these findings indicated any adverse effect of treatment of treatment with TS-ED 532 at dietary concentrations of 1500, 6000 or 25000 ppm in either generation.

Histopathology:
Histopathological examination was performed on behavioral offspring and is described in the developmental neurotoxicity part described in section7.9.1 Neurotoxicity - "2-generation reproduction/developmental neurotoxicity study main study - developmental neurotoxicity part".

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 342 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 493 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

Results: F2 generation

Effect levels (F2)

1

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

>= 1 342 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

>= 1 493 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1. Animals allocated to treatment groups for the two generation investigation.

Treatment Group	Group Number	Dietary Concentration (ppm)	Animal Numbers
			F0 generation		F1 generation
			Male	Female	Male	Female
TS-ED 532 (Control)	3	0	28 (1-28)	28 (141-168)	24 (281-304)	24 (401-424)
TS-ED 532 (Low)	2	1500	28 (29-56)	28 (169-196)	24 (305-328)	24 (425-448)
TS-ED 532 (Intermediate)	4	6000	28 (57-84)	28 (197-224)	24 (329-352)	24 (449-472)
TS-ED 532 (High)	1	25000*	28 (85-112)	28 (225-252)	24 (353-376)	24 (473-496)
DEHP (Positive Control)	5	10000	28 (113-140)	28 (253-280)	24 (377-400)	24 (497-503,  505-507  509-522)#

*Animals initially received 15000 ppm rising to 20000 ppm and finally 25000 ppm during the maturation phase of each generation.

# Females 504 and 508 died during the early stages of the F₁ generation and were replaced with animals 521 and 522.

Table 2. Animals allocated to treatment groups for the investigation of developmental neurotoxicity.

Treatment Group TS-ED 532	Group Number	Dietary Concentration (ppm)	Number of Offspring
			Pre-weaning		Post weaning
			F0-F1	F1-F2	F0-F1A	F0-F1B
Control	3	0	13M, 13F	11M, 10F	26M, 26F	26M, 26F
Low	2	1500	13M, 12F	12M, 12F	25M, 24F	25M, 24F
Intermediate	4	6000	13M, 13F	11M, 10F	26M, 26F	26M, 26F
High	1	25000	12M, 11F	12M, 12F	22M, 22F	19M, 19F

A: Offspring allocated to majority of post-weaning assessments, B: Offspring allocated to water maze on Days 61-62 of age.

Table3. The dietary concentrations resulted in the follwoing mean achieved dosages.

Dietary Concentration	F0 Males (mg/kg bw/day)	F0 Females (mg/kg bw/day)
		Maturation	Gestation	Lactation
1500 ppm TS-ED 532	82	106	101	231
6000 ppm TS-ED 532	324	431	411	919
25000 ppm TS-ED 532	1159	1392	1665	3544

Dietary Concentration	F1 Males (mg/kg bw/day)	F1 Females (mg/kg bw/day)
		Maturation	Gestation	Lactation
1500 ppm TS-ED 532	109	135	108	238
6000 ppm TS-ED 532	435	540	434	918
25000 ppm TS-ED 532	1342	1493	1697	3596

Dietary Concentration	Generation	Males (mg/kg/day)	F0Females (mg/kg/day)
			Maturation	Gestation	Lactation
10000 ppm DEHP (positive control group)	F0	579	705	689	1131
10000 ppm DEHP (positive control group)	F1	1141	1136

Table 4. Organ Weights for Female Offspring - Group Mean Values - F₀- F₁Generation

Group/Dietary Concentration		Bodyweight (g) at Terminal Kill	Absolute Organ Weight (g)				Relative Organ Weight (%) D
			Brain	Spleen	Thymus	Uterus	Brain	Spleen	Thymus	Uterus
3 - Control 0 ppm	mean	43.8	1.4243	0.1861	0.1779	0.0923	3.3261	0.4226	0.4095	0.2111
	sd	8.0	0.0783	0.0576	0.0479	0.0265	0.4395	0.0615	0.0827	0.0322
	n	24	25	25	25	25	24	24	24	24
2 - Low TS-ED 532 1500 ppm	mean	41.1	1.3749	0.1644	0.1644	0.0877	3.4238	0.3899	0.3984	0.2119
	sd	6.8	0.1089	0.0526	0.0456	0.0318	0.5663	0.0879	0.0824	0.0601
	n	24	24	23	24	24	24	23	24	24
4 - Inter TS-ED 532 6000 ppm	mean	42.6	1.3728	0.1642	0.1800	0.0831	3.2829	0.3762	0.4200	0.1931*
	sd	7.2	0.0934	0.0589	0.0510	0.0274	0.3810	0.0764	0.0798	0.0456
	n	25	25	25	25	25	25	25	25	25
1 - High TS-ED 532 25000 ppm	mean	41.0	1.3755	0.1485*	0.1764	0.1242	3.4449	0.3553**	0.4293	0.2967
	sd	7.8	0.0816	0.0513	0.0522	0.1746	0.5422	0.0719	0.0877	0.4169
	n	23	23	23	23	23	23	23	23	23

D= % of bodyweight, * = P≤0.05, **= P≤0.01, ***= P≤0.001

Table 5.Organ Weights for Female Offspring - Group Mean Values - F₁ -F₂

Group/Dietary Concentration		Bodyweight (g) at Terminal Kill	Absolute Organ Weight (g)				Relative Organ Weight (%) D
			Brain	Spleen	Thymus	Uterus	Brain	Spleen	Thymus	Uterus
3 - Control 0 ppm	mean	44.6	1.4174	0.1868	0.1841	0.0539	3.2663	0.4096	0.4114	0.1210
	sd	8.4	0.0682	0.0646	0.0464	0.0113	0.5079	0.0799	0.0510	0.0158
	n	21	21	21	21	21	21	21	21	21
2 - Low TS-ED 532 1500 ppm	mean	40.8	1.3677	0.1451*	0.1586	0.0536	3.4250	0.3507*	0.3937	0.1324
	sd	6.8	0.0736	0.0443	0.0363	0.0182	0.4671	0.0527	0.0808	0.0426
	n	24	24	24	24	24	24	24	24	24
4 - Inter TS-ED 532 6000 ppm	mean	41.3	1.3702	0.1534	0.1820	0.0513	3.3866	0.3656	0.4417	0.1225
	sd	6.6	0.0613	0.0493	0.0389	0.0212	0.4465	0.0713	0.0627	0.0348
	n	22	22	22	22	22	22	22	22	22
1 - High TS-ED 532 25000 ppm	mean	40.7	1.3814	0.1370**	0.1710	0.0517	3.5429	0.3257***	0.4152	0.1276
	sd	9.9	0.0903	0.0590	0.0532	0.0242	0.6535	0.0748	0.0526	0.0489
	n	24	24	24	24	24	24	24	24	24

D= % of bodyweight, *= P≤0.05, **= P≤0.01, ***= P≤0.001

Due to the limited amount of space in this section, summary tables from the final study report has been attached as a separate PDF file ("2 -generation reproduction and developmental neurotoxicity study - Summary Tables"). The PDF file is attached as background material in the section below.

Applicant's summary and conclusion

Conclusions:

Overall, TS-ED 532 was found not to have effects on reproduction, pre natal development and post-natal development of the rat when administered to two successive generations, including no endocrine disrupting effect using ano-genital distance and nipple count as effect parameters. Furthermore, TS-ED 532 was found not to induce any developmental neurotoxicity in the offspring. The animals were exposed to 1500, 6000 or 25000 ppm TS-ED 532 in the feed. NOEL of TS-ED 532 for adult toxicity and reproduction over the two generations was 25000 ppm, giving exposure of at least 1000 mg/kg bw/day throughout all of the study . Other than a decrease in spleen weights for female offspring, the NOEL of TS-ED 532 for offspring development was 25000 ppm and this dosage represents a clear NOAEL for offspring development. NOEL of TS-ED 532 for offspring survival and growth, and for developmental neurotoxicity was 25000 ppm. The mean achieved dosages at this exposure level were 1159 mg/kg bw/day (F0 male), 2200 mg/kg bw/day (F0 female), 1320 mg/kg bw/day (F1 male) and 2262 (F1 female). The lowest identified NOAEL at exposure level 25000 ppm was ≥ 1159 mg/kg bw/day (F0 male).

Executive summary:

The study performed represents a combination study design specifically taking into consideration the requirements of the OECD guideline 416 and 426. A combination study design was chosen based on the requirements of using the minimum number of test animals. The study was performed to investigate the effects of TS-ED 532 on reproduction, pre-natal development and post-natal development of the rat when administered to two successive generations. Measurement of antiandrogen endpoints (nipple retention at PND 12-15 and ano-genital distance at PND 1) was included to evaluate a possible endocrine disrupting effect. The study also investigated the potential of TS-ED 532 to cause developmental neurotoxicity in the offspring. An optionally 3-generation reproduction study was to be performed if effects on reproduction was identified earlier in the study. The study also incorporated a positive control group for endocrine disruption (ano-genital distance, nipple retention and sexual development) using DEHP throughout the F0 generation and until sexual maturation of the F1 offspring.

TS-ED 532 was administered orally by dietary admixture to three groups each of twenty-eight male and twenty-eight female F₀ Generation Sprague-Dawley strain rats and subsequently twenty-four male and twenty-four female F₁Generation rats, over a maturation period of at least 10 weeks, and throughout mating, gestation and lactation for each generation. Animals allocated to low and intermediate dosage groups received intended dietary inclusion levels of 1500 or 6000 ppm, respectively. In each generation, animals allocated to the high dosage group initially received an intended dietary inclusion level of 15000 ppm, rising to 20000 ppm and then 25000 ppm during the maturation period and being sustained at the higher inclusion level until termination. A further group of twenty-eight male and twenty-eight female F₀ Generation rats and subsequently twenty-four male and twenty-four female F₁ Generation animals received basal diet, containing an amount of Arachis Oil equivalent to TS-ED 532 added to the high dosage diet, over the same treatment period to serve as a control.

The positive control group induced the expected effects on ano-genital distance, nipple counts and delays in attainment of sexual maturation, thereby validating the study.

Overall, TS-ED 532 was found not to have effects on reproduction, pre natal development and post-natal development of the rat when administered to two successive generations, including no endocrine disrupting effect using ano-genital distance and nipple count as effect parameters. Furthermore, TS-ED 532 was found not to induce any developmental neurotoxicity in the offspring. The animals were exposed to 1500, 6000 or 25000 ppm TS-ED 532 in the feed. NOEL of TS-ED 532 for adult toxicity and reproduction over the two generations was 25000 ppm, giving exposure of at least 1000 mg/kg bw/day throughout all of the study . Other than a decrease in spleen weights for female offspring, the NOEL of TS-ED 532 for offspring development was 25000 ppm and this dosage represents a clear NOAEL for offspring development. NOEL of TS-ED 532 for offspring survival and growth, and for developmental neurotoxicity was 25000 ppm. The mean achieved dosages at this exposure level were 1159 mg/kg bw/day (F0 male), 2200 mg/kg bw/day (F0 female), 1320 mg/kg bw/day (F1 male) and 2262 (F1 female). The lowest identified NOAEL at exposure level 25000 ppm was ≥ 1159 mg/kg bw/day (F0 male).