6-(2-chloro-6-cyano-4-nitrophenylazo)-4-methoxy-3-[N-(methoxycarbonylmethyl)-N-(1-methoxycarbonylethyl)amino]acetanilide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 02 September 2014 and 07 April 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

See below

Principles of method if other than guideline:

None

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were
obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the
animals were examined for signs of ill-health or injury. The animals were acclimatized for
eight days during which time their health status was assessed. A total of ninety six animals
(forty eight males and forty eight females) were accepted into the study. At the start of treatment
the males weighed 301 to 356g, the females weighed 190 to 223g, and were approximately
twelve weeks old.
Initially, all animals were housed in groups of four in solid floor polypropylene cages with
stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the
pairing phase, the animals were transferred to polypropylene grid floor cages suspended over
trays lined with absorbent paper on a one male: one female basis. Following evidence of
successful mating, the males were returned to their original cages. Mated females were housed
individually during gestation and lactation in solid floor polypropylene cages with stainless steel
mesh lids and softwood flakes.
The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad
Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Mains drinking water was
supplied from polycarbonate bottles attached to the cage. Environmental enrichment was
provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd.,
Cheshire, UK) except for paired animals and mated females during the final week of gestation
and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation
and lactation. The diet, drinking water, bedding and environmental enrichment were considered
not to contain any contaminant at a level that might have affected the purpose or integrity of the
study.
The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd.,
Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen
air changes per hour and the low intensity fluorescent lighting was controlled to give twelve
hours continuous light and twelve hours darkness. Environmental conditions were continuously
monitored by a computerized system, and print-outs of hourly temperatures and humidities are
included in the study records. The Study Plan target ranges for temperature and relative
humidity were 22 ± 3 °C and 50 ± 20% respectively; Short term deviations from these targets
were considered not to have affected the purpose or integrity of the study; see deviations from
Study Plan.
The animals were randomly allocated to treatment groups using a stratified body weight
randomization procedure and the group mean body weights were then determined to ensure
similarity between the treatment groups. The cage distribution within the holding rack was also
randomized. The animals were uniquely identified within the study by an ear punching system
routinely used in these laboratories.

Justification
The rat was selected for this study as it is a readily available rodent species historically used in
safety evaluation studies and is acceptable to appropriate regulatory authorities.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

Test Item Preparation
For the purpose of this study the test item was prepared at the appropriate concentrations as a
suspension in Arachis oil BP. The stability and homogeneity of the test item formulations were
determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the
formulations to be stable for at least 9 days. Formulations were therefore prepared weekly and
stored at approximately 4 °C in the dark.
Samples of the test item formulation were taken and analyzed for concentration of FAT 41024/A
TE at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate
that the prepared formulations were within ± 11% of the nominal concentration.

Details on mating procedure:

On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a
maximum of fourteen days.
Following evidence of mating (designated as Day 0 post coitum) the males were returned
to their original cages and females were transferred to individual cages.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Introduction
The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

Test item
The test item described in the main part of the study was also used as the analytical standard.

Analytical procedure
Stock solutions of test item in acetonitrile were prepared for external calibration. An aliquot, approximately 0.1 g of test item was accurately weighed into a 100 mL volumetric flask and brought to volume with acetonitrile to yield a solution with a concentration of 1 mg/mL.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.

Analysis of samples
The formulations received were extracted with acetonitrile. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with acetonitrile this was then ultra-sonicated for 30 minutes and centrifuged at 4500 rpm for 10 minutes. Where necessary, sample solutions were further diluted with acetonitrile to achieve the working concentration.

Preparation of accuracy samples
Samples of Arachis Oil BP were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. There samples were then prepared for analysis as the test samples.

Preparation of linearity standards
A range of standard solutions were prepared in acetonitrile from a stock solution of 1.079 mg/mL by serial dilution covering the concentration range 0 to 0.1619 mg/mL.

Instrumental setup
HPLC: Agilent Technologies 1100 or LC36) 1200, incorporating autosampler and workstation.
Column: Gemini C18 100A 5 µ (100 x 4.6 mm id)
Column Temp: 40 °C
Mobile phase:
Eluent A: Water
Eluent B: Acetonitrile
Time %B
0 10
5 90
10 90
Flow rate: 1 mL/min
UV detector wavelength: 254 nm
Injection volume: 10 µL
Retention time: ~ 7 mins

Study samples and storage
Representative samples were dispatched to the analytical laboratories internally (under ambient conditions) and stored at room temperature until analysis.

Results
Validation of analytical method
Specificity
The control dose samples and an analysed solvent blank showed no significant interfering response at the retention time of the test item. The standard solutions contained a peak specific for the test item whose area changed accordingly with known concentration; hence the specificity of the method by retention time was confirmed.

Linearity
The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. The data was found to have linear correlation within the calibration range. The R2 fit of the calibration curve to the data was 0.9999 and was considered to be acceptable.

Accuracy
The fortified samples of Arachis Oil BP were found to have a recovery value of ± 10 % of the fortification.

Test item formulations
The formulations investigated during the study were found to comprise test item in the range of 100 – 111%.
The test item was found to be stable in the formulations when kept for 9 days in the refrigerator (4 °C) due to results which met the variation limit of 10 % from the time-zero mean.
In conclusion, the results indicate the accurate use of the test item and Arachis Oil BP as vehicle during this study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was proven.

Discussion
The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven to be suitable for use.

Duration of treatment / exposure:

Males: 43 days
Females: Up to 65 days (to Day 5 post partum)
Any female which did not produce a pregnancy or a live litter was terminated after Day 25 post coitum.

Frequency of treatment:

Daily

Details on study schedule:

Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated daily at the appropriate
dose level throughout the study (except for females during parturition where applicable).
The first day of dosing was designated as Day 1 of the study.
ii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a
maximum of fourteen days.
iii. Following evidence of mating (designated as Day 0 post coitum) the males were returned
to their original cages and females were transferred to individual cages.
iv. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post
partum. Litter size, offspring weight and sex, surface righting and clinical signs were
also recorded during this period.
v. The male dose groups were killed and examined macroscopically on Day 43.
vi. At Day 5 post partum, all females and surviving offspring were killed and examined
macroscopically.

Remarks:

Doses / Concentrations:
50, 150 and 500 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Twelve male and twelve female per group

Control animals:

yes, concurrent vehicle

Details on study design:

Procedure
The test item was administered daily by gavage using a stainless steel cannula attached to a
disposable plastic syringe. Control animals were treated in an identical manner with 6 mL/kg of
Arachis oil BP.
The volume of test and control item administered to each animal was based on the most recent
scheduled body weight and was adjusted at weekly intervals.

Positive control:

None

Parental animals: Observations and examinations:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change
immediately before dosing, soon after dosing, and one hour after dosing during the working
week (except for females during parturition were applicable). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males
until termination and weekly for females until pairing. During the pairing phase females were
weighed daily until mating was confirmed. Body weights were then recorded for females on
Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults
until pairing. This was continued for males after the mating phase. For females showing
evidence of mating, food consumption was recorded for the periods covering post coitum Days
0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the
lactation period (Days 1-4).
Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males
and for females during the pre-pairing phase. Due to offspring growth and milk production for
lactation, food efficiency for females could not be accurately calculated during gestation and
lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.

Reproductive Performance
Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to
fourteen days. 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 estrus 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 (Day 0 of gestation) and the males were subsequently
returned to their original holding cages. Mated females were housed individually during the
period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as
late as possible during the normal working day) around the period of expected parturition.
Observations were carried out at approximately 08:30 and as late as possible at weekends. The
following was recorded 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

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

No data

Litter observations:

On completion of parturition (Day 0 post partum), the number of live and dead offspring was
recorded. Offspring were individually identified within each litter by tattoo 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 Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated
retrospectively from this data)

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Postmortem examinations (parental animals):

Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by
exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable
barbiturate agent followed by exsanguination on Day 5 post partum. Any females which failed to
achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.
For all females, the uterus was examined for signs of implantation and the number of uterine
implantations in each horn was recorded. This procedure was enhanced; as necessary, by
staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora
lutea were also counted.
All adult animals and offspring, including those dying during the study, were subjected to a full
external and internal examination, and any macroscopic abnormalities were recorded.


All offspring, including those dying during the study, were subjected to a full
external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The epididymides and testes were removed from terminal kill adult males dissected free from fat
and weighed before fixation.

Histopathology
Samples of the following tissues were preserved from all animals from each dose group, in
buffered 10% formalin, except where stated:

Coagulating gland, Prostate, Epididymides ♦, Seminal vesicles, Ovaries, Testes ♦, Mammary gland, Uterus/Cervix, Pituitary, Vagina.
♦ preserved in Modified Davidsons fluid

All tissues were dispatched to the histology processing Test Site (Propath UK Ltd, Willow
Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal
Investigator: N Fower). The tissues from control and 500 mg/kg bw/day dose group animals,
and any animals which did not achieve a pregnancy were prepared as paraffin blocks, sectioned
at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent
microscopic examination. In addition, sections of testes from all control and 500 mg/kg bw/day
males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, 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,
multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
Any cell or stage-specificity of testicular findings was noted.

Postmortem examinations (offspring):

Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent.
All offspring, including those dying during the study, were subjected to a full
external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

See below

Reproductive indices:

Reproductive Indices
Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:

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

ii. Fertility Indices
For each group the following were calculated:

Mating index (%) = (number of animals mated / number of animals paired) x 100

Pregnancy index (%) = number of pregnant females / number of animals mated) x 100

Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:

i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii. Parturition Index
The following was calculated for each group:
Parturition index (%) = (number of females delivering live offspring / number of pregnant females) x 100

Offspring viability indices:

The standard unit of assessment was considered to be the litter, therefore values were first
calculated for each litter and the group mean was calculated using their individual litter values.
Group mean values included all litters reared to termination (Day 5 of age).

i. Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for
each female/litter as follows:

Pre–implantation loss (%) = ((Number of corpora lutea - Number of implantation sites) / Number of corpora lutea) x 100

Post–implantation loss (%) = ((Number of implantation sites - Total number of offspring born) / Number of implantation sites) x 100

ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:

Live birth index (%) = number of offspring alive on Day 1 / number of offspring born) x 100
Viability index (%) = number of offspring alive on Day 4 / number of offspring alive on Day 1) x 100

iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:

(number of male offspring / total number of offspring) x 100

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See results

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See results

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Mortality
There were no unscheduled deaths.

Clinical Observations
There were no clinical signs at any dose levels considered to be related to toxicity of the test
item.
At 500 mg/kg bw/day, findings were confined to episodes of increased post dosing salivation for
animals of both sexes between Days 9 and 19. Observations of this nature are commonly
observed following the oral administration of an unpalatable or slightly irritant test item
formulation; in isolation and at the frequency observed these findings were considered not to
indicate toxicity from the test item. All treated animals showed instances of fur stained by the
test item. Such observations are often reported following administration of a colored test item
and simply represents normal grooming behavior and dispersal of the coloration over external fur
surfaces.
One male treated with 50 mg/kg bw/day was observed to have a small wound on the forelimb
which later developed into scab formation during Week 5. This was a physical injury and
unrelated to treatment.

Body Weight
Body weight gains in 500 mg/kg bw/day males were statistically significantly reduced during the
first two weeks of treatment. Recovery was evident thereafter however overall body weight gain
was lower when compared to controls.
Females treated with 500 mg/kg bw/day showed a statistically significant reduction in body
weight gain during week one of treatment. Recovery was evident thereafter.

No such effects were detected in animals of either sex treated with 150 or 50 mg/kg bw/day.

Food Consumption
There were no treatment-related effects on dietary intake noted for males during the study or for
females during the pre-pairing, gestation or lactation phases of the study.
At 500 mg/kg bw/day food conversion efficiency for either sex was marginally lower than
controls. This correlated with the initial reductions in body weight gain seen at this level and
recovery was evident thereafter; therefore this was considered not to represent an adverse effect
of treatment.

Water Consumption
Daily visual inspection of water bottles did not indicate any overt differences in water
consumption at 50, 150 or 500 mg/kg bw/day.

Reproductive Performance
Mating
No treatment-related effects were detected in mating performance.

Fertility
There were no treatment-related differences in fertility.
At 150 mg/kg bw/day one female did not achieve pregnancy following positive evidence of
mating. No histopathological correlates were evident in the reproductive organs of the female or
the corresponding male partner to cause the infertility, therefore in the absence of a similar effect
at 500 mg/kg bw/day this was considered unrelated to treatment. One female treated with
150 mg/kg bw/day had corpora lutea and implantation sites but failed to give birth to any live
offspring. In isolation and in the absence of a similar effect at 500 mg/kg bw/day the intergroup
difference was considered unrelated to treatment.

Gestation Length
There were no differences in gestation lengths. The distribution for the majority of treated
females was comparable to controls. Gestation lengths were between 22 and 24 days.

Pathology
Necropsy
There were no macroscopic abnormalities detected in terminal kill animals.

Organ Weights
No treatment-related changes were evident in the organ weights measured. Statistical analysis of
the data did not reveal any significant intergroup differences.

Histopathology
There were no treatment-related microscopic abnormalities detected in the tissue and organs
examined.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

In total, 12, 12, 10 and 12 females from 0 (control), 50, 150 and 500 mg/kg bw/day
(respectively) dose groups gave birth to a live litter and successfully reared young to Day 5 of
age. The following assessment of litter response is based on all litters reared to termination on
Day 5 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability
Group mean corpora lutea and implantation counts, litter size, implantation losses, survival
indices and sex ratio are given in Tables 8 to 10. Individual data are given in Appendices 6 to 8.
There were no treatment-related effects detected for corpora lutea, implantation counts, pre or
post implantation losses, litter size or litter viability for treated animals when compared to
controls. There were no intergroup differences in sex ratio (percentage male offspring) for litters
from treated groups when compared to controls. Statistical analysis of the data did not reveal any
significant intergroup differences.
A slight increase in pre-implantation loss was evident at 500 mg/kg bw/day however this was
considered to be the result of one female which showed a higher loss. The mean value excluding
this female was comparable to controls therefore the intergroup difference was considered to
reflect normal biological variation rather than an effect of treatment.

Offspring Growth and Development
There were no toxicologically significant effects detected.
Statistical analysis of the litter, offspring weights or surface righting reflex data did not reveal
any significant intergroup differences.
A slight reduction in litter weight was evident at 500 mg/kg bw/day on Days 1 and 4 post
partum. This was considered to be the result of one litter which only comprised three offspring.
Mean values excluding this litter were comparable to controls therefore the intergroup
differences were considered to reflect normal biological variation rather than an effect of
treatment.
There were no obvious clinical signs of toxicity detected for offspring when compared to
controls. The incidental clinical signs detected throughout the control and treated groups,
consisting of cold, killed in extremis due to physical injury, missing or found dead and
generalized bruising were considered to be low incidence findings observed in offspring in
studies of this type and were considered unrelated to test item toxicity.

Pathology
Necropsy
No treatment-related macroscopic abnormalities were detected for interim death or terminal kill
offspring. The incidental findings observed were those occasionally observed in reproductive
studies of this type and were considered to be unrelated to toxicity of the test item.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Reproductive effects observed:

not specified

Discussion

The oral administration of FAT 41024/A TE to rats for a period of up to forty-two days for males and up to eight weeks for females (including two weeks pre-mating, gestation and early lactation period) at levels of 50, 150 and 500 mg/kg bw/day was well tolerated. There were no clinical signs to indicate systemic toxicity, although animals of either sex treated with 500 mg/kg bw/day showed episodes of excessive salivation post dose and sporadic instances of blue fur staining due to the test item was observed in animals from all test groups. Such observations are often reported following oral administration of an unpalatable or slightly irritant test item formulation and of a colored test item and represents normal grooming behavior, dispersal of the coloration over the external fur surface. Therefore these observations are considered of no toxicological significance. At 500 mg/kg bw/day, reduced body weight gains were observed in males during the first and second week of treatment and during the first week of treatment for females. Body weight gains for either sex were comparable to controls thereafter although overall gain for males was lower when compared to controls at the end of the treatment period. No effect on food intake was evident however a slight reduction in food conversion efficiency was evident during the first week of treatment for either sex at 500 mg/kg bw/day. This was considered to be a result of the fluctuating body weight gain at this level. Therefore these intergroup differences were considered to represent an initial insult of the test item but not an overall adverse effect of treatment. In relation to reproduction/developmental toxicity, there were no effects on mating performance or fertility or any evidence of offspring developmental effects at any level.

Conclusions:

The oral administration of FAT 41024/A TE to rats by gavage, at dose levels of 50, 150 and
500 mg/kg bw/day, was well tolerated. Based on limited evaluations for systemic toxicity the
‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 500 mg/kg bw/day. The
‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 500 mg/kg
bw/day.

Executive summary:

Introduction

The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, and provides an initial hazard assessment for effect on reproduction. The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995). This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 50, 150 and 500 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP). Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy or a live litter was terminated after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

Results

Adult Responses

Mortality

There were no unscheduled deaths.

Clinical Observations

There were no clinical signs at any dose levels considered to be related to toxicity of the test item.

Body Weight

Body weight gains in 500 mg/kg bw/day males were lower than control males during the first two weeks of treatment. Recovery was evident thereafter however overall body weight gain was lower when compared to controls. A reduction in body weight gain was also evident in females treated with 500 mg/kg bw/day during Week 1 of treatment. Recovery was evident thereafter. No such effects were detected in animals of either sex treated with 150 mg/kg bw/day or in animals of either sex treated with 50 mg/kg bw/day.

Food Consumption

No adverse effects were detected for food consumption or food conversion efficiency.

Water Consumption

Visual inspection of water residues did not indicate any effect of treatment on water intake throughout the study.

Reproductive Performance

Mating

No treatment-related effects were detected in mating performance.

Fertility

There were no treatment-related differences in fertility.

Gestation Length

There were no differences in gestation lengths. The distribution for treated females was compared to controls. Gestation lengths were between 22 and 24 days.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum was comparable to controls. Sex ratio in treated litters was comparable to controls.

Offspring Growth and Development

Offspring body weight gains and litter weights on Days 1 and 4 post partum were unaffected by treatment. Surface righting reflex in treated litters was comparable to controls. The clinical signs apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of material treatment on offspring development.

Pathology

Necropsy There were no treatment related macroscopic abnormalities detected.

Organ Weights

No treatment-related changes were evident in the organ weights measured.

Histopathology

There was no evidence of any treatment-related histopathological changes in the organs and tissues examined.

Conclusion

The oral administration of FAT 41024/A TE to rats by gavage, at dose levels of 50, 150 and 500 mg/kg bw/day, was well tolerated. Based on limited evaluations for systemic toxicity the ‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 500 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 500 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Study Klimisch 1.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, and provides an initial hazard assessment for effect on reproduction.

The test item was administered by gavage to three groups, each of twelve male and twelve female rats, for up to eight weeks (including a two week pre-pairing phase, pairing gestation and early lactation for females), at dose levels of 50, 150 and 500 mg/kg bw /day.

During the experiment, they were no unscheduled deaths, no clinical signs at any dose levels considered to be related to toxicity of the test item.

The body weight gains in 500 mg/kg bw/day males was were lower than control male during the first two weeks of treatment. A reduction of body weight gain was also evident in females treated with 500 mg/kg bw/day during week 1. Recovery for both sex was evident thereafter (for males overall body weight gain was lower when compared to controls). Reproductive performance, fertility, gestation length and litter response were not affected. Offspring growth and development were also unaffected by treatment.

The oral administration of the test item to rats by gavage was well tolerated. Based on limited evaluations for systemic toxicity the "No Observed Adverse Effect Level" (NOAEL) was considered to be 500 mg/kg bw/day. The "No Observed Effect Level" (NOEL) for reproductive toxicity was considered to be 500 mg/kg bw/day.

Short description of key information:
Based on limited evaluations for systemic toxicity the "No Observed Adverse Effect Level" (NOAEL) was considered to be 500 mg/kg bw/day. The "No Observed Effect Level" (NOEL) for reproductive toxicity was considered to be 500 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
Only this study is available.

Effects on developmental toxicity

Description of key information

Based on limited evaluations for systemic toxicity the "No Observed Adverse Effect Level" (NOAEL) was considered to be 500 mg/kg bw/day. The "No Observed Effect Level" (NOEL) for reproductive toxicity was considered to be 500 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 02 September 2014 and 07 April 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, No.421: “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995)

Deviations:

yes

Remarks:

See below

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Animals and Animal Husbandry
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were
obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the
animals were examined for signs of ill-health or injury. The animals were acclimatized for
eight days during which time their health status was assessed. A total of ninety six animals
(forty eight males and forty eight females) were accepted into the study. At the start of treatment
the males weighed 301 to 356g, the females weighed 190 to 223g, and were approximately
twelve weeks old.
Initially, all animals were housed in groups of four in solid floor polypropylene cages with
stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the
pairing phase, the animals were transferred to polypropylene grid floor cages suspended over
trays lined with absorbent paper on a one male: one female basis. Following evidence of
successful mating, the males were returned to their original cages. Mated females were housed
individually during gestation and lactation in solid floor polypropylene cages with stainless steel
mesh lids and softwood flakes.
The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad
Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Mains drinking water was
supplied from polycarbonate bottles attached to the cage. Environmental enrichment was
provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd.,
Cheshire, UK) except for paired animals and mated females during the final week of gestation
and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation
and lactation. The diet, drinking water, bedding and environmental enrichment were considered
not to contain any contaminant at a level that might have affected the purpose or integrity of the
study.
The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd.,
Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen
air changes per hour and the low intensity fluorescent lighting was controlled to give twelve
hours continuous light and twelve hours darkness. Environmental conditions were continuously
monitored by a computerized system, and print-outs of hourly temperatures and humidities are
included in the study records. The Study Plan target ranges for temperature and relative
humidity were 22 ± 3 °C and 50 ± 20% respectively; Short term deviations from these targets
were considered not to have affected the purpose or integrity of the study; see deviations from
Study Plan.
The animals were randomly allocated to treatment groups using a stratified body weight
randomization procedure and the group mean body weights were then determined to ensure
similarity between the treatment groups. The cage distribution within the holding rack was also
randomized. The animals were uniquely identified within the study by an ear punching system
routinely used in these laboratories.

Justification
The rat was selected for this study as it is a readily available rodent species historically used in
safety evaluation studies and is acceptable to appropriate regulatory authorities.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

Test Item Preparation
For the purpose of this study the test item was prepared at the appropriate concentrations as a
suspension in Arachis oil BP. The stability and homogeneity of the test item formulations were
determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the
formulations to be stable for at least 9 days. Formulations were therefore prepared weekly and
stored at approximately 4 °C in the dark.
Samples of the test item formulation were taken and analyzed for concentration of FAT 41024/A
TE at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate
that the prepared formulations were within ± 11% of the nominal concentration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Introduction
The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

Test item
The test item described in the main part of the study was also used as the analytical standard.

Analytical procedure
Stock solutions of test item in acetonitrile were prepared for external calibration. An aliquot, approximately 0.1 g of test item was accurately weighed into a 100 mL volumetric flask and brought to volume with acetonitrile to yield a solution with a concentration of 1 mg/mL.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.

Analysis of samples
The formulations received were extracted with acetonitrile. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with acetonitrile this was then ultra-sonicated for 30 minutes and centrifuged at 4500 rpm for 10 minutes. Where necessary, sample solutions were further diluted with acetonitrile to achieve the working concentration.

Preparation of accuracy samples
Samples of Arachis Oil BP were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. There samples were then prepared for analysis as the test samples.

Preparation of linearity standards
A range of standard solutions were prepared in acetonitrile from a stock solution of 1.079 mg/mL by serial dilution covering the concentration range 0 to 0.1619 mg/mL.

Instrumental setup
HPLC: Agilent Technologies 1100 or LC36) 1200, incorporating autosampler and workstation.
Column: Gemini C18 100A 5 µ (100 x 4.6 mm id)
Column Temp: 40 °C
Mobile phase:
Eluent A: Water
Eluent B: Acetonitrile
Time %B
0 10
5 90
10 90
Flow rate: 1 mL/min
UV detector wavelength: 254 nm
Injection volume: 10 µL
Retention time: ~ 7 mins

Study samples and storage
Representative samples were dispatched to the analytical laboratories internally (under ambient conditions) and stored at room temperature until analysis.

Results
Validation of analytical method
Specificity
The control dose samples and an analysed solvent blank showed no significant interfering response at the retention time of the test item. The standard solutions contained a peak specific for the test item whose area changed accordingly with known concentration; hence the specificity of the method by retention time was confirmed.

Linearity
The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. The data was found to have linear correlation within the calibration range. The R2 fit of the calibration curve to the data was 0.9999 and was considered to be acceptable.

Accuracy
The fortified samples of Arachis Oil BP were found to have a recovery value of ± 10 % of the fortification.

Test item formulations
The formulations investigated during the study were found to comprise test item in the range of 100 – 111%.
The test item was found to be stable in the formulations when kept for 9 days in the refrigerator (4 °C) due to results which met the variation limit of 10 % from the time-zero mean.
In conclusion, the results indicate the accurate use of the test item and Arachis Oil BP as vehicle during this study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was proven.

Discussion
The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven to be suitable for use.

Details on mating procedure:

On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a
maximum of fourteen days.
Following evidence of mating (designated as Day 0 post coitum) the males were returned
to their original cages and females were transferred to individual cages.

Duration of treatment / exposure:

Males: 43 days
Females: Up to 65 days (to Day 5 post partum)
Any female which did not produce a pregnancy or a live litter was terminated after Day 25 post coitum.

Frequency of treatment:

Daily

Duration of test:

Males: 43 days
Females: Up to 65 days (to Day 5 post partum)
Any female which did not produce a pregnancy or a live litter was terminated after Day 25 post coitum.

No. of animals per sex per dose:

Twelve male and twelve female per group

Control animals:

yes, concurrent vehicle

Details on study design:

Procedure
The test item was administered daily by gavage using a stainless steel cannula attached to a
disposable plastic syringe. Control animals were treated in an identical manner with 6 mL/kg of
Arachis oil BP.
The volume of test and control item administered to each animal was based on the most recent
scheduled body weight and was adjusted at weekly intervals.

Maternal examinations:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change
immediately before dosing, soon after dosing, and one hour after dosing during the working
week (except for females during parturition were applicable). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males
until termination and weekly for females until pairing. During the pairing phase females were
weighed daily until mating was confirmed. Body weights were then recorded for females on
Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults
until pairing. This was continued for males after the mating phase. For females showing
evidence of mating, food consumption was recorded for the periods covering post coitum Days
0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the
lactation period (Days 1-4).
Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males
and for females during the pre-pairing phase. Due to offspring growth and milk production for
lactation, food efficiency for females could not be accurately calculated during gestation and
lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.

Reproductive Performance
Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to
fourteen days. 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 estrus 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 (Day 0 of gestation) and the males were subsequently
returned to their original holding cages. Mated females were housed individually during the
period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as
late as possible during the normal working day) around the period of expected parturition.
Observations were carried out at approximately 08:30 and as late as possible at weekends. The
following was recorded 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

Necropsy
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by
exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable
barbiturate agent followed by exsanguination on Day 5 post partum. Any females which failed to
achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.

All adult animals and offspring, including those dying during the study, were subjected to a full
external and internal examination, and any macroscopic abnormalities were recorded.


All offspring, including those dying during the study, were subjected to a full
external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The epididymides and testes were removed from terminal kill adult males dissected free from fat
and weighed before fixation.

Histopathology
Samples of the following tissues were preserved from all animals from each dose group, in
buffered 10% formalin, except where stated:

Coagulating gland, Prostate, Epididymides ♦, Seminal vesicles, Ovaries, Testes ♦, Mammary gland, Uterus/Cervix, Pituitary, Vagina.
♦ preserved in Modified Davidsons fluid

All tissues were dispatched to the histology processing Test Site (Propath UK Ltd, Willow
Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal
Investigator: N Fower). The tissues from control and 500 mg/kg bw/day dose group animals,
and any animals which did not achieve a pregnancy were prepared as paraffin blocks, sectioned
at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent
microscopic examination. In addition, sections of testes from all control and 500 mg/kg bw/day
males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, 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,
multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
Any cell or stage-specificity of testicular findings was noted.

Ovaries and uterine content:

For all females, the uterus was examined for signs of implantation and the number of uterine
implantations in each horn was recorded. This procedure was enhanced; as necessary, by
staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora
lutea were also counted.

Fetal examinations:

On completion of parturition (Day 0 post partum), the number of live and dead offspring was
recorded. Offspring were individually identified within each litter by tattoo 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 Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated
retrospectively from this data)

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Necropsy
Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent.
All offspring, including those dying during the study, were subjected to a full
external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

See below

Indices:

See below

Historical control data:

No data

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality
There were no unscheduled deaths.

Clinical Observations
There were no clinical signs at any dose levels considered to be related to toxicity of the test
item.
At 500 mg/kg bw/day, findings were confined to episodes of increased post dosing salivation for
animals of both sexes between Days 9 and 19. Observations of this nature are commonly
observed following the oral administration of an unpalatable or slightly irritant test item
formulation; in isolation and at the frequency observed these findings were considered not to
indicate toxicity from the test item. All treated animals showed instances of fur stained by the
test item. Such observations are often reported following administration of a colored test item
and simply represents normal grooming behavior and dispersal of the coloration over external fur
surfaces.
One male treated with 50 mg/kg bw/day was observed to have a small wound on the forelimb
which later developed into scab formation during Week 5. This was a physical injury and
unrelated to treatment.

Body Weight
Body weight gains in 500 mg/kg bw/day males were statistically significantly reduced during the
first two weeks of treatment. Recovery was evident thereafter however overall body weight gain
was lower when compared to controls.
Females treated with 500 mg/kg bw/day showed a statistically significant reduction in body
weight gain during week one of treatment. Recovery was evident thereafter.

No such effects were detected in animals of either sex treated with 150 or 50 mg/kg bw/day.

Food Consumption
There were no treatment-related effects on dietary intake noted for males during the study or for
females during the pre-pairing, gestation or lactation phases of the study.
At 500 mg/kg bw/day food conversion efficiency for either sex was marginally lower than
controls. This correlated with the initial reductions in body weight gain seen at this level and
recovery was evident thereafter; therefore this was considered not to represent an adverse effect
of treatment.

Water Consumption
Daily visual inspection of water bottles did not indicate any overt differences in water
consumption at 50, 150 or 500 mg/kg bw/day.

Reproductive Performance
Mating
No treatment-related effects were detected in mating performance.

Fertility
There were no treatment-related differences in fertility.
At 150 mg/kg bw/day one female did not achieve pregnancy following positive evidence of
mating. No histopathological correlates were evident in the reproductive organs of the female or
the corresponding male partner to cause the infertility, therefore in the absence of a similar effect
at 500 mg/kg bw/day this was considered unrelated to treatment. One female treated with
150 mg/kg bw/day had corpora lutea and implantation sites but failed to give birth to any live
offspring. In isolation and in the absence of a similar effect at 500 mg/kg bw/day the intergroup
difference was considered unrelated to treatment.

Gestation Length
There were no differences in gestation lengths. The distribution for the majority of treated
females was comparable to controls. Gestation lengths were between 22 and 24 days.

Pathology
Necropsy
There were no macroscopic abnormalities detected in terminal kill animals.

Organ Weights
No treatment-related changes were evident in the organ weights measured. Statistical analysis of
the data did not reveal any significant intergroup differences.

Histopathology
There were no treatment-related microscopic abnormalities detected in the tissue and organs
examined.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
In total, 12, 12, 10 and 12 females from 0 (control), 50, 150 and 500 mg/kg bw/day
(respectively) dose groups gave birth to a live litter and successfully reared young to Day 5 of
age. The following assessment of litter response is based on all litters reared to termination on
Day 5 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability
Group mean corpora lutea and implantation counts, litter size, implantation losses, survival
indices and sex ratio are given in Tables 8 to 10. Individual data are given in Appendices 6 to 8.
There were no treatment-related effects detected for corpora lutea, implantation counts, pre or
post implantation losses, litter size or litter viability for treated animals when compared to
controls. There were no intergroup differences in sex ratio (percentage male offspring) for litters
from treated groups when compared to controls. Statistical analysis of the data did not reveal any
significant intergroup differences.
A slight increase in pre-implantation loss was evident at 500 mg/kg bw/day however this was
considered to be the result of one female which showed a higher loss. The mean value excluding
this female was comparable to controls therefore the intergroup difference was considered to
reflect normal biological variation rather than an effect of treatment.

Offspring Growth and Development
There were no toxicologically significant effects detected.
Statistical analysis of the litter, offspring weights or surface righting reflex data did not reveal
any significant intergroup differences.
A slight reduction in litter weight was evident at 500 mg/kg bw/day on Days 1 and 4 post
partum. This was considered to be the result of one litter which only comprised three offspring.
Mean values excluding this litter were comparable to controls therefore the intergroup
differences were considered to reflect normal biological variation rather than an effect of
treatment.
There were no obvious clinical signs of toxicity detected for offspring when compared to
controls. The incidental clinical signs detected throughout the control and treated groups,
consisting of cold, killed in extremis due to physical injury, missing or found dead and
generalized bruising were considered to be low incidence findings observed in offspring in
studies of this type and were considered unrelated to test item toxicity.

Pathology
Necropsy
No treatment-related macroscopic abnormalities were detected for interim death or terminal kill
offspring. The incidental findings observed were those occasionally observed in reproductive
studies of this type and were considered to be unrelated to toxicity of the test item.

Abnormalities:

not specified

Developmental effects observed:

not specified

Discussion

The oral administration of FAT 41024/A TE to rats for a period of up to forty-two days for males and up to eight weeks for females (including two weeks pre-mating, gestation and early lactation period) at levels of 50, 150 and 500 mg/kg bw/day was well tolerated. There were no clinical signs to indicate systemic toxicity, although animals of either sex treated with 500 mg/kg bw/day showed episodes of excessive salivation post dose and sporadic instances of blue fur staining due to the test item was observed in animals from all test groups. Such observations are often reported following oral administration of an unpalatable or slightly irritant test item formulation and of a colored test item and represents normal grooming behavior, dispersal of the coloration over the external fur surface. Therefore these observations are considered of no toxicological significance. At 500 mg/kg bw/day, reduced body weight gains were observed in males during the first and second week of treatment and during the first week of treatment for females. Body weight gains for either sex were comparable to controls thereafter although overall gain for males was lower when compared to controls at the end of the treatment period. No effect on food intake was evident however a slight reduction in food conversion efficiency was evident during the first week of treatment for either sex at 500 mg/kg bw/day. This was considered to be a result of the fluctuating body weight gain at this level. Therefore these intergroup differences were considered to represent an initial insult of the test item but not an overall adverse effect of treatment. In relation to reproduction/developmental toxicity, there were no effects on mating performance or fertility or any evidence of offspring developmental effects at any level.

Conclusions:

The oral administration of FAT 41024/A TE to rats by gavage, at dose levels of 50, 150 and
500 mg/kg bw/day, was well tolerated. Based on limited evaluations for systemic toxicity the
‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 500 mg/kg bw/day. The
‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 500 mg/kg
bw/day.

Executive summary:

Introduction

The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, and provides an initial hazard assessment for effect on reproduction. The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995). This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods…….

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 50, 150 and 500 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP). Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy or a live litter was terminated after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

Results…….

Adult Responses

Mortality

There were no unscheduled deaths.

Clinical Observations

There were no clinical signs at any dose levels considered to be related to toxicity of the test item.

Body Weight

Body weight gains in 500 mg/kg bw/day males were lower than control males during the first two weeks of treatment. Recovery was evident thereafter however overall body weight gain was lower when compared to controls. A reduction in body weight gain was also evident in females treated with 500 mg/kg bw/day during Week 1 of treatment. Recovery was evident thereafter. No such effects were detected in animals of either sex treated with 150 mg/kg bw/day or in animals of either sex treated with 50 mg/kg bw/day.

Food Consumption

No adverse effects were detected for food consumption or food conversion efficiency.

Water Consumption

Visual inspection of water residues did not indicate any effect of treatment on water intake throughout the study.

Reproductive Performance

Mating

No treatment-related effects were detected in mating performance.

Fertility

There were no treatment-related differences in fertility.

Gestation Length

There were no differences in gestation lengths. The distribution for treated females was compared to controls. Gestation lengths were between 22 and 24 days.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum was comparable to controls. Sex ratio in treated litters was comparable to controls.

Offspring Growth and Development

Offspring body weight gains and litter weights on Days 1 and 4 post partum were unaffected by treatment. Surface righting reflex in treated litters was comparable to controls. The clinical signs apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of material treatment on offspring development.

Pathology

Necropsy There were no treatment related macroscopic abnormalities detected.

Organ Weights

No treatment-related changes were evident in the organ weights measured.

Histopathology

There was no evidence of any treatment-related histopathological changes in the organs and tissues examined.

Conclusion

The oral administration of FAT 41024/A TE to rats by gavage, at dose levels of 50, 150 and 500 mg/kg bw/day, was well tolerated. Based on limited evaluations for systemic toxicity the ‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 500 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 500 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Study Klimisch 1.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Justification for selection of Effect on developmental toxicity: via oral route:
Only this study is available.

Toxicity to reproduction: other studies

Additional information

The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, and provides an initial hazard assessment for effect on reproduction.

The test item was administered by gavage to three groups, each of twelve male and twelve female rats, for up to eight weeks (including a two week pre-pairing phase, pairing gestation and early lactation for females), at dose levels of 50, 150 and 500 mg/kg bw /day.

During the experiment, they were no unscheduled deaths, no clinical signs at any dose levels considered to be related to toxicity of the test item.

The body weight gains in 500 mg/kg bw/day males was were lower than control male during the first two weeks of treatment. A reduction of body weight gain was also evident in females treated with 500 mg/kg bw/day during week 1. Recovery for both sex was evident thereafter (for males overall body weight gain was lower when compared to controls). Reproductive performance, fertility, gestation length and litter response were not affected. Offspring growth and development were also unaffected by treatment.

The oral administration of the test item to rats by gavage was well tolerated. Based on limited evaluations for systemic toxicity the "No Observed Adverse Effect Level" (NOAEL) was considered to be 500 mg/kg bw/day. The "No Observed Effect Level" (NOEL) for reproductive toxicity was considered to be 500 mg/kg bw/day.

Justification for classification or non-classification

The available data on the reproduction/developmental toxicity screening test of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.