Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The NOAEL for reproductive toxicity was determined to be 1000 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Test start date: 03 September 2014; Test end date: 28 April 2015; Study completion date: 06 May 2015.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
See below.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Identification: FAT 40543/A TE
Physical State/Appearance: Red solid
Purity: 99.4 %
Batch Number: TV RN 196-200
Label : Lot.Nr.: TV RN196-200 VD 24.04.2019
Date Received: 26 August 2014
Storage Conditions: Room temperature, in the dark
Expiry Date: 24 April 2019
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 six 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 310 to 346 g; the females weighed 200 to 235 g, and were approximately twelve weeks old. Initially, all animals were housed in groups of three 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 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 humidity 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:
CMC (carboxymethyl cellulose)
Remarks:
1%
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 1 % Carboxy methylcellulose. 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 13 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 40543/A TE at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 7 % of the nominal concentration. 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 8 mL/kg of 1 % Carboxy methylcellulose. 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.
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 two peaks.

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

Analytical procedure
Preparation of standard solutions
Stock solutions of test item in acetonitrile were prepared for external standard 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. Aliquots of this stock standard solution were used to prepare working standard solutions in acetonitrile with a concentration of 0.01 mg/mL. On each occasion standard solutions derived from two stock standard solutions were used for calculation.

Analysis of samples
Formulations received were diluted with acetonitrile. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with acetonitrile; this was then shaken and sonicated to dissolve. Where necessary, sample solutions were further diluted with acetonitrile to achieve the working concentration.

Preparation of accuracy samples
Samples of 1 % CMC were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were prepared for analysis as for the test samples.

Preparation of linearity standard
A range of standard solutions were prepared in acetonitrile from a stock solution of 1.001 mg/mL by serial dilution covering the concentration range 0 to 0.02002 mg/mL.

Instrument setup
HPLC: Agilent Technologies 1200, incorporating autosampler and workstation.
Column: Gemini C18 100A 5 µ (100 x 4.6 mm id)
Mobile phase:
Eluent A: water
Eluent B: Acetonitrile
Time %B
0 10
5 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 analyzed 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 concentrations; hence the specificity of the method by retention time was conformed.

Linearity
The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard cocnentrations. The data was found to have a linear correlation within the calibration range. The R2 fit of the calibration curve to the data was 0.999 and considered to be acceptable.

Accuracy
The fortified samples of 1 % CMC 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 the 93 to 101 % and thus the required content limit of ± 10 % with reference to the nominal content was met. The test item was found to be stable in the formulations when kept for 13 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 1 % CMC as vehicle during this study. The formulations were found to be homogenously prepared and sufficient formulation stability under storage conditions was proven.
Duration of treatment / exposure:
Adult males were terminated on Day 43 or 44
Adult females and offspring were terminated on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or 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 or 44.
vi.;
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose group
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Mid dose group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose group
No. of animals per sex per dose:
12 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
- Rationale for animal assignment: Random
Positive control:
None
Parental animals: Observations and examinations:
Observations
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 and at weekends (except for females during parturition where 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 oestrus 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):
Necropsy
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43 or 44. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent. Any females which failed to achieve pregnancy 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.


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 (females only), Uterus/Cervix, Pituitary, Vagina,
♦ preserved in Modified Davidsons fluid

All tissues were dispatched to the histology processing Test Site (Huntingdon Life Sciences Ltd., Eye Research Centre, Eye, Suffolk IP23 7PX) for processing (Principal Investigator: J Schofield). The tissues from control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, 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 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined; see deviations from the Study Plan. 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. Microscopic examination was conducted by the Study Pathologist (W Henderson) and a peer review was performed by test site (P. Millar at Peter Millar Associates Ltd., 3 Queen Charlotte Lane, Edinburgh, EH6 6AY).
Postmortem examinations (offspring):
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:
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 animas 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 site – 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
Mortality:
no mortality 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. Sporadic instances of red fur staining from the test item were observed in animals of either sex receiving 1000 mg/kg bw/day from Day 6 of dosing and one male given 300 mg/kg bw/day from Day 8 of dosing. In addition, cages of both sexes from all test groups were observed to have instances of red stained bedding. Such observations are often reported following administration of a colored test item and represents normal excretion of the colored compound and, following normal grooming behavior, dispersal of the coloration over the external fur surface. One male treated with 300 mg/kg bw/day was observed to have pilo erection on Day 34 and hunched posture on Days 34 and 35; due to the isolated nature of the incidence and in the absence of similar observations at 1000 mg/kg bw/day this was considered not to be of toxicological importance.

Body Weight
At 1000 and 300 mg/kg bw/day, lower body weight gain was notable for both sexes during the first week of treatment when compared with controls, attaining statistical significance in females (p<0.05) at both dosages during the pre-pairing phase. However, improvement of body weight gain was apparent for both sexes thereafter. Pregnant females at 1000 and 300 mg/kg bw/day, showed slight reductions in group mean body weights and attained statistical significance (p<0.05 or p<0.01) when compared with controls during gestation. This was considered to reflect the group mean body weights at the start of gestation after the initial lower gains during the first week of treatment. As body weight gains during gestation and lactation were comparable to controls and a true dose related response was not evident, the intergroup differences were considered not to be of any toxicological significance. No such effects were detected in animals of either sex treated with 100 mg/kg bw/day.

Food Consumption: There were no toxicological significant effects detected for food consumption or food conversion efficiency throughout the study. At 1000 and 300 mg/kg bw/day, food consumption and food conversion efficiency was very slightly lower when compared to controls during the first week of treatment for males, this would reflect the body weight gain reduction apparent for these animals. Improvement for these animals was evident thereafter. Food conversion efficiency was reduced during the first week of treatment for females treated with 1000 and 300 mg/kg bw/day. Food intake was unaffected in these females and the reduced food conversion efficiency was considered to reflect the lower body weight gains seen in these females.

Water Consumption: Daily visual inspection of water bottles did not indicate any overt differences in water consumption at 100, 300 or 1000 mg/kg bw/day.

Reproductive Performance
Mating: There were no treatment-related effects detected in mating performance.

Fertility: There were no treatment-related differences in fertility. One female treated with 1000 mg/kg bw/day and one female treated with 100 mg/kg bw/day did not achieve pregnancy following positive evidence of mating. At the histopathogical assessment, the 1000 mg/kg bw/day female had inflammation of the uterus, but both females appeared to be cycling normally and the corresponding male partners appeared normal. The intergroup differences were therefore considered to be incidental.

Gestation Length: There were no treatment-related differences in gestation lengths. The distribution for treated females was comparable to controls. Gestation lengths were between 22 and 23½ days.

Necropsy: No toxicologically significant macroscopic abnormalities were detected. Macroscopic examination revealed one female treated with 1000 mg/kg bw/day to have a brown fluid filled uterus and cervix, and one male treated with 300 mg/kg bw/day, to have a significantly enlarged and fluid filled (left) kidney and pale (left) seminal vesicle. In the absence of any treatement-related microscopic changes these intergroup differences are consistent with normally expected low incidence findings in laboratory maintained rats, and were considered not to be related to test item toxicity.

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 findings found at histopathological examination. One female treated with 1000 mg/kg bw/day had inflammation of the uterus which correlated with the brown fluid filled horns detected at necropsy. One male treated with 300 mg/kg bw/day was found to have hydronephrosis in the kidneys which would account for the significantly enlarged kidney found at necropsy. Both of these findings were of an isolated incidence and therefore were considered not to be of toxicological significance.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related adverse effects were observed
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
not examined
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
Litter Responses: In total, 12, 11, 12 and 11 females from 0 (control), 100, 300 and 1000 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: There was no adverse effect of maternal treatment on the numbers of corpora lutea and implantations, pre and post implantation losses, litter size at birth/Day 1 and subsequent offspring survival to Day 4 of age at 100, 300 or 1000 mg/kg bw/day. Sex ratio (percentage male offspring) for the offspring was similar in all groups and did not indicate any selective effect of maternal treatment on survival for either sex.

Offspring Growth and Development: There were no toxicologically significant effects detected.
Offspring weights for both sexes were marginally lower on Days 1 and 4 post partum for litters where females were treated with 1000 mg/kg bw/day. Litter weights on Days 1 and 4 post partum were also slightly reduced at this level. Offspring body weight gain however at 1000 mg/kg bw/day between Days 1 and 4 post partum were comparable to controls and actually exceeded litters treated with 100 mg/kg bw/day. Statistical analysis of litter data did not reveal any significant intergroup differences therefore the slight intergroup differences were considered to reflect normal biological variation and not an effect of treatment. No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, cold, pale, weak, no milk in stomach, missing, found dead, cannibalized, generalized bruising or darkened abdominal region were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.

Necropsy: Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence or distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 100, 300 or 1000 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related adverse effects were observed
Key result
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
The oral administration of FAT 40543/A TE to rats by gavage, at dose levels of 100, 300 and 1000 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 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 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 seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (1 % Carboxy methylcellulose). 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 or 44, followed by the termination of all females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on or after Day 25post 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 1000 mg/kg bw/day males were notably lower than that of the control males after one week of treatment. Males receiving 300 mg/kg bw/day also showed slightly a lower body weight gain during the first week of treatment. Recovery was evident thereafter. Body weight gains during the first week of maturation of females receiving 300 and 1000 mg/kg bw/day were lower than that of the control. Recovery was evident thereafter. Body weight development in both sexes at 100 mg/kg bw/day remained unaffected by treatment.

 

Food Consumption

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

 

Water Consumption

There was no noticeable adverse effects on water consumption by visual inspection.

 

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 treatment-related differences in gestation lengths. The distribution for treated females was comparable to controls. Gestation lengths were between 22 and 23½ days.

 

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

Of the litters born, litter size at birth and subsequently on Days 1 and 4post partumwas 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 4post partumwere unaffectedby treatment. Surface righting in treated litters was comparable to controls. The clinical signs apparent for offspring on the study were typical for the age observed.

 

Pathology

Necropsy

Macroscopic findings for adults and offspring did not indicate any adverse effect of treatment.

 

Organ Weights

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

 

Histopathology

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

 

Conclusion

The oral administration of FAT 40543/A to rats by gavage, at dose levels of 100, 300 and 1000 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 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day. 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
High quality GLP study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Currently no study to assess the reproductive and/or developmental toxicity of Disperse Yellow 114 is available. However, the read-across substance Disperse Yellow 246 was evaluated in a study performed according to OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995).  In this study, 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 seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (1 % Carboxy methylcellulose). 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 or 44, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed. There were no unscheduled deaths. There were no clinical signs at any dose levels considered to be related to toxicity of the test item. Body weight gains in 1000 mg/kg bw/day males were notably lower than that of the control males after one week of treatment. Males receiving 300 mg/kg bw/day also showed slightly a lower body weight gain during the first week of treatment. Recovery was evident thereafter. Body weight gains during the first week of maturation of females receiving 300 and 1000 mg/kg bw/day were lower than that of the control. Recovery was evident thereafter. Body weight development in both sexes at 100 mg/kg bw/day remained unaffected by treatment. No adverse effects were detected in food consumption or food conversion efficiency. There was no noticeable adverse effects on water consumption by visual inspection. No treatment-related effects were detected in mating performance, fertility or gestation lengths. The distribution for treated females was comparable to controls. Gestation lengths were between 22 and 23½ days. 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 body weight gains and litter weights on Days 1 and 4post partumwere unaffectedby treatment. Surface righting in treated litters was comparable to controls. The clinical signs apparent for offspring on the study were typical for the age observed. Macroscopic findings for adults and offspring did not indicate any adverse effect of treatment. No treatment-related changes were evident in the organ weights measured. There was no evidence of treatment-related histopathological changes in the organs and tissues examined. Based on the above findings, the ‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive as well as developmental toxicity was considered to be 1000 mg/kg bw/day. 

Effects on developmental toxicity

Description of key information

The NOAEL for developmental toxicity was determined to be 1000 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:
Test start date: 03 September 2014; Test end date: 28 April 2015; Study completion date: 06 May 2015.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
See below
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Identification: FAT 40543/A TE
Physical State/Appearance: Red solid
Purity: 99.4%
Batch Number : TV RN 196-200
Label : Lot.Nr.: TV RN196-200 VD 24.04.2019 Raumtemperatur menge : 500g
Date Received : 26 August 2014
Storage Conditions : Room temperature, in the dark
Expiry Date : 24 April 2019
Species:
rat
Strain:
Wistar
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 six 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 310 to 346 g; the females weighed 200 to 235g, and were approximately twelve weeks old.

Initially, all animals were housed in groups of three 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 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 humidity 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:
CMC (carboxymethyl cellulose)
Remarks:
1%
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 1% Carboxy methylcellulose. 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 13 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 40543/A TE at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 7% of the nominal concentration.

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 8 mL/kg of 1% Carboxy methylcellulose.

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.
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 two peaks.

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

Analytical procedure
Preparation of standard solutions
Stock solutions of test item in acetonitrile were prepared for external standard 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. Aliquots of this stock standard solution were used to prepare working standard solutions in acetonitrile with a concentration of 0.01 mg/mL. On each occasion standard solutions derived from two stock standard solutions were used for calculation.

Analysis of samples
Formulations received were diluted with acetonitrile. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with acetonitrile; this was then shaken and sonicated to dissolve. Where necessary, sample solutions were further diluted with acetonitrile to achieve the working concentration.

Preparation of accuracy samples
Samples of 1 % CMC were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were prepared for analysis as for the test samples.

Preparation of linearity standard
A range of standard solutions were prepared in acetonitrile from a stock solution of 1.001 mg/mL by serial dilution covering the concentration range 0 to 0.02002 mg/mL.

Instrument setup
HPLC: Agilent Technologies 1200, incorporating autosampler and workstation.
Column: Gemini C18 100A 5 µ (100 x 4.6 mm id)
Mobile phase:
Eluent A: water
Eluent B: Acetonitrile
Time %B
0 10
5 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 analyzed 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 concentrations; hence the specificity of the method by retention time was conformed.

Linearity
The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard cocnentrations. The data was found to have a linear correlation within the calibration range. The R2 fit of the calibration curve to the data was 0.999 and considered to be acceptable.

Accuracy
The fortified samples of 1 % CMC 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 the 93 to 101 % and thus the required content limit of ± 10 % with reference to the nominal content was met.
The test item was found to be stable in the formulations when kept for 13 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 1 % CMC as vehicle during this study. The formulations were found to be homogenously prepared and sufficient formulation stability under storage conditions was proven.
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:
Adult males were terminated on Day 43 or 44.
Adult females and offspring were terminated on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose group
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Mid dose group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose group
No. of animals per sex per dose:
12 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
- Rationale for animal assignment: Random
Maternal examinations:
Observations
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 and at weekends (except for females during parturition where 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 oestrus 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
Fetal examinations:
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
All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
See results
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Details on results:
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.
Sporadic instances of red fur staining from the test item were observed in animals of either sex receiving 1000 mg/kg bw/day from Day 6 of dosing and one male given 300 mg/kg bw/day from Day 8 of dosing. In addition, cages of both sexes from all test groups were observed to have instances of red stained bedding. Such observations are often reported following administration of a colored test item and represents normal excretion of the colored compound and, following normal grooming behavior, dispersal of the coloration over the external fur surface.
One male treated with 300 mg/kg bw/day was observed to have pilo erection on Day 34 and hunched posture on Days 34 and 35; due to the isolated nature of the incidence and in the absence of similar observations at 1000 mg/kg bw/day this was considered not to be of toxicological importance.


Body Weight
At 1000 and 300 mg/kg bw/day, lower body weight gain was notable for both sexes during the first week of treatment when compared with controls, attaining statistical significance in females (p<0.05) at both dosages during the pre-pairing phase. However, improvement of body weight gain was apparent for both sexes thereafter.
Pregnant females at 1000 and 300 mg/kg bw/day, showed slight reductions in group mean body weights and attained statistical significance (p<0.05 or p<0.01) when compared with controls during gestation. This was considered to reflect the group mean body weights at the start of gestation after the initial lower gains during the first week of treatment. As body weight gains during gestation and lactation were comparable to controls and a true dose related response was not evident, the intergroup differences were considered not to be of any toxicological significance.
No such effects were detected in animals of either sex treated with 100 mg/kg bw/day.

Food Consumption: There were no toxicological significant effects detected for food consumption or food conversion efficiency throughout the study.
At 1000 and 300 mg/kg bw/day, food consumption and food conversion efficiency was very slightly lower when compared to controls during the first week of treatment for males, this would reflect the body weight gain reduction apparent for these animals. Improvement for these animals was evident thereafter. Food conversion efficiency was reduced during the first week of treatment for females treated with 1000 and 300 mg/kg bw/day. Food intake was unaffected in these females and the reduced food conversion efficiency was considered to reflect the lower body weight gains seen in these females.

Water Consumption: Daily visual inspection of water bottles did not indicate any overt differences in water consumption at 100, 300 or 1000 mg/kg bw/day.

Reproductive Performance
Mating: There were no treatment-related effects detected in mating performance.

Fertility: There were no treatment-related differences in fertility.
One female treated with 1000 mg/kg bw/day and one female treated with 100 mg/kg bw/day did not achieve pregnancy following positive evidence of mating. At the histopathogical assessment, the 1000 mg/kg bw/day female had inflammation of the uterus, but both females appeared to be cycling normally and the corresponding male partners appeared normal. The intergroup differences were therefore considered to be incidental.

Gestation Length: There were no treatment-related differences in gestation lengths. The distribution for treated females was comparable to controls. Gestation lengths were between 22 and 23½ days.

Necropsy: No toxicologically significant macroscopic abnormalities were detected.

Macroscopic examination revealed one female treated with 1000 mg/kg bw/day to have a brown fluid filled uterus and cervix, and one male treated with 300 mg/kg bw/day, to have a significantly enlarged and fluid filled (left) kidney and pale (left) seminal vesicle. In the absence of any treatement-related microscopic changes these intergroup differences are consistent with normally expected low incidence findings in laboratory maintained rats, and were considered not to be related to test item toxicity.

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 findings found at histopathological examination.
One female treated with 1000 mg/kg bw/day had inflammation of the uterus which correlated with the brown fluid filled horns detected at necropsy.
One male treated with 300 mg/kg bw/day was found to have hydronephrosis in the kidneys which would account for the significantly enlarged kidney found at necropsy.
Both of these findings were of an isolated incidence and therefore were considered not to be of toxicological significance.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Mating
There were no treatment-related effects detected in mating performance.

Fertility
There were no treatment-related differences in fertility.
One female treated with 1000 mg/kg bw/day and one female treated with 100 mg/kg bw/day did not achieve pregnancy following positive evidence of mating. At the histopathogical assessment, the 1000 mg/kg bw/day female had inflammation of the uterus, but both females appeared to be cycling normally and the corresponding male partners appeared normal. The intergroup differences were therefore considered to be incidental.

Gestation Length
There were no treatment-related differences in gestation lengths. The distribution for treated females was comparable to controls. Gestation lengths were between 22 and 23½ days.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No treatment related adverse effects were observed
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Litter Responses
In total, 12, 11, 12 and 11 females from 0 (control), 100, 300 and 1000 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
There was no adverse effect of maternal treatment on the numbers of corpora lutea and implantations, pre and post implantation losses, litter size at birth/Day 1 and subsequent offspring survival to Day 4 of age at 100, 300 or 1000 mg/kg bw/day. Sex ratio (percentage male offspring) for the offspring was similar in all groups and did not indicate any selective effect of maternal treatment on survival for either sex.

Offspring Growth and Development
There were no toxicologically significant effects detected. Offspring weights for both sexes were marginally lower on Days 1 and 4 post partum for litters where females were treated with 1000 mg/kg bw/day. Litter weights on Days 1 and 4 post partum were also slightly reduced at this level. Offspring body weight gain however at 1000 mg/kg bw/day between Days 1 and 4 post partum were comparable to controls and actually exceeded litters treated with 100 mg/kg bw/day. Statistical analysis of litter data did not reveal any significant intergroup differences therefore the slight intergroup differences were considered to reflect normal biological variation and not an effect of treatment.

No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisiting of small size, cold, pale, weak, no milk in stomach, missing, found dead, cannibalized, generalized brusing or darkened abdominal region were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.

Necropsy
Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence or distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 100, 300 or 1000 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related adverse effects were observed
Abnormalities:
no effects observed
Developmental effects observed:
no

The oral administration of FAT 40543/A TE to rats for a period of up to forty-three days for males and up to seven weeks for females (including two weeks pre-mating, gestation and early lactation period) at dose levels of 100, 300 or 1000 mg/kg bw/day was well tolerated.

There were no clinical signs to indicate systemic toxicity, although oral administration of the formulated test item was associated with sporadic instances of red fur staining due to the test item in animals of either sex receiving 1000 mg/kg bw/day and one male given 300 mg/kg bw/day. All test groups were also observed to have instances of red stained bedding. Such observations are often reported following the oral administration of a colored test item and represents normal excretion of the colored compound and, following normal grooming behavior, dispersal of the coloration over the external fur surface. Therefore these observations are considered of no toxicological significance.

Lower body weight gains were observed for animals of either sex during the first week of treatment at 1000 and 300 mg/kg bw/day. Improvement was evident for both sexes thereafter, therefore the 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 dose levels.

Conclusions:
The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was considered to be 1000 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 seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (1% Carboxy methylcellulose).

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 or 44, followed by the termination of all females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on or 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 1000 mg/kg bw/day males were notably lower than that of the control males after one week of treatment. Males receiving 300 mg/kg bw/day also showed slightly a lower body weight gain during the first week of treatment. Recovery was evident thereafter. Body weight gains during the first week of maturation of females receiving 300 and 1000 mg/kg bw/day were lower than that of the control. Recovery was evident thereafter. Body weight development in both sexes at 100 mg/kg bw/day remained unaffected by treatment.

Food Consumption

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

Water Consumption

There was no noticeable adverse effects on water consumption by visual inspection.

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 treatment-related differences in gestation lengths. The distribution for treated females was comparable to controls. Gestation lengths were between 22 and 23½ 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 partumwas 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 partumwere unaffected by treatment. Surface righting in treated litters was comparable to controls. The clinical signs apparent for offspring on the study were typical for the age observed.

Pathology

Necropsy

Macroscopic findings for adults and offspring did not indicate any adverse effect of treatment.

Organ Weights

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

Histopathology

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

Conclusion

The oral administration of FAT 40543/A TE to rats by gavage, at dose levels of 100, 300 and 1000 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 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive and developmetal toxicity was considered to be 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
High quality GLP study
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

As discussed in the 'additional information' section of effects on fertility above, the source substance Disperse Yellow 246 had no adverse effects on developmental process or functions, hence, the NOAEL for developmental toxicity was determined to be 1000 mg/kg bw/day.

Justification for classification or non-classification

Based on the available data, Disperse Yellow 114 does not warrant the classification as per the CLP (EU regulation No. 1272/2008) criteria.

Additional information