Methyl 2-[(1,5-dihydro-3-methyl-5-oxo-1-phenyl-4H-pyrazol-4-ylidene)ethylidene]-1,3,3-trimethylindoline-5-carboxylate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 08 February 2017 Experimental Completion Date: 12 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 2-[(1,5-dihydro-3-methyl-5-oxo-1-phenyl-4H-pyrazol-4-ylidene)ethylidene]-1,3,3-trimethylindoline-5-carboxylate

EC Number:

227-217-5

EC Name:

Methyl 2-[(1,5-dihydro-3-methyl-5-oxo-1-phenyl-4H-pyrazol-4-ylidene)ethylidene]-1,3,3-trimethylindoline-5-carboxylate

Cas Number:

5718-26-3

Molecular formula:

C25H25N3O3

IUPAC Name:

methyl 1,3,3-trimethyl-2-[2-(3-methyl-5-oxo-1-phenyl-1,5-dihydro-4H-pyrazol-4-ylidene)ethylidene]indoline-5-carboxylate

Constituent 2

Reference substance name:

4-[(1,5-dihydro-3-methyl-5-oxo-1-phenyl-4H-pyrazol-4-ylidene)methyl]-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one

EC Number:

225-184-1

EC Name:

4-[(1,5-dihydro-3-methyl-5-oxo-1-phenyl-4H-pyrazol-4-ylidene)methyl]-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one

Cas Number:

4702-90-3

Molecular formula:

C21H18N4O2

IUPAC Name:

4,4'-methylylidenebis(5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one)

Test material form:

solid

Details on test material:

Macrolex Orange R, Batch CHA 001

Specific details on test material used for the study:

Identification: Macrolex Orange R
Physical State/Appearance: Red Powder
Date Received: 03 June 2016
Storage Conditions: Stored in darkness; under ambient conditions, used/ formulated in the light
Expiry Date: 18 February 2018
No correction for purity was made.

Test animals

Species:

rat

Strain:

other: Wistar Han™:RccHan™:WIST

Details on species / strain selection:

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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for nineteen days during which time their health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment. 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 283 to 359g and were approximately eleven weeks old. The females weighed 194 to 231g and were approximately fourteen weeks old.

Animal Care and Husbandry
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, Envigo RMS (UK) Limited 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 Envigo Research Limited, 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; there were no deviations from these targets.
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.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least eighteen days. Formulations were therefore prepared weekly and stored at approximately 4 °C in the dark.



.

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:

Samples of the test item formulation were taken on four occasions and analyzed for concentration of Macrolex Orange R at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 99 to 107% of the nominal concentration.

Duration of treatment / exposure:

approximately six weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females)

Frequency of treatment:

daily

Details on study schedule:

Chronological Sequence of Study
i. Males and females were housed for a suitable acclimatization period which allowed at least two weeks of pre-treatment vaginal smears to be performed for females enabling the exclusion of females not showing appropriate estrous cycling.
ii. 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.
iii. For the 14 days prior to pairing pre-pairing vaginal smears were performed and assessed for females.
iv. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
v. 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.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 13 post partum. Litter size, offspring weight and sex, ano-genital distance and visible nipple counts (male offspring) and clinical signs were also recorded during this period.
vii. On day 4 post partum, where possible, blood sampling was performed on two randomly allocated offspring from each litter in order to obtain serum samples.
viii. The male dose groups were killed and examined macroscopically on Day 44 or 45.
ix. On Day 13 post partum, where possible, blood sampling to produce serum samples for assessment of thyroid hormones was performed on two randomly selected offspring (one male and one female) per litter. Where possible, a further two randomly selected offspring (one male and one female) per litter were sampled to produce plasma samples. Thyroid samples were also retained from one male and one female from each litter where litter sizes allowed. All offspring were killed and examined externally, where external observations were detected an internal necropsy was performed.
x. All females were killed on Day 14 post partum and examined macroscopically. A vaginal smear was also performed for all females in the morning of the day of necropsy. Any female which did not produce a pregnancy was also killed and examined macroscopically around the same time as littering females. In addition, blood samples to produce both serum and plasma were taken from all animals at termination for possible thyroid hormone analysis.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 males and 12 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were chosen in consultation with the sponsor, and based on the results of previous toxicity work including a Twenty-Eight Day Repeated Dose Oral (Gavage) Toxicity Study in the Rat (Envigo Study Number DX05TC). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

Examinations

Parental animals: Observations and examinations:

Serial Observations
General Observations/Measurements
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 (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, 4 7 and 14 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 for the periods covering post partum days 1-4, 4-7 and 7-14.
Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males through-out the study period (with the exception of the mating phase) 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.
Intergroup differences did not indicate any need for more formal gravimetric measurements.

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 0830 and as late as possible at weekends and public holidays. 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):

Vaginal smears were taken daily for females throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.

Sperm parameters (parental animals):

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.

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, 4, 7 and 14 post partum
iii. Sex of offspring on Days 1, 4 and 13 post partum
iv. Clinical condition of offspring from birth to Day 13 post partum
v. Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data)


Physical Development
All live offspring were assessed for ano-genital distance on Day 1 post partum. Additionally, visible nipple count was performed for all male offspring on Day 13 post partum.

Postmortem examinations (parental animals):

Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 44 or 45. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 14 post partum. Any females which failed to achieve pregnancy or produce a litter were killed around the same time as littering females.
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).
All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. As the test item was of colored appearance, any discoloration was documented to judge local and systemic bioavailability.

Thyroid Hormone Assessment
Where possible, blood samples taken to produce serum were allowed to clot, centrifuged and the serum from each blood sample was stored frozen at lower than -60 °C for possible evaluation of thyroid hormone. Blood samples to produce plasma were collected into K2EDTA, centrifuged, and the plasma from each blood sample stored frozen at lower than -60 ºC for possible evaluation of thyroid hormones. Samples were taken as follows:
Serum and plasma samples were taken from all adult males and all adult females at termination.
All serum samples were shipped to the test site (Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS) for serum analysis, frozen, packed in dry ice. The serum from adult males and Day 13 offspring were analyzed for Thyroxine (T4) under the supervision of the Prinicpal Investigator (H Bose).

Organ Weights
The epididymides, testes, seminal vesicles (with coagulation gland) and prostate were removed from terminal kill adult males, dissected free from fat and weighed before fixation. Thyroid/parathyroid were dissected free from fat for terminal kill animals from both sexes, and weighed after being placed in fixative (partial fixation).

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

Cowpers Glands
Pituitary
Epididymides ♦
Prostate
Glans Penis
Seminal vesicles (and coagulating gland)
Gross lesions
Testes ♦
LABC (levator ani-bulbocavernous) Muscle
Thyroid/Parathyroid
Mammary gland
Uterus/Cervix (and oviducts)
Ovaries
Vagina
♦ preserved in Modified Davidsons fluid

Staining in the adipose tissue, stomach, cecum, liver and kidneys was observed on one or more occasion in treated animals. These tissues were therefore retained from five control males and five control females, but were not processed at histopathology. Stained tissues observed in treated animals were retained as gross lesions, but were also not processed following discussions with the Sponsor.
All tissues were dispatched to the histology processing Test Site (Envigo CRS Limited, Eye Research Centre, Eye, Suffolk, IP23 7PX) for processing (Principal Investigator: D Roberts). The epididymides, ovaries, and testes from control and 1000 mg/kg bw/day dose group animals, any animals which failed to achieve a pregnancy and any abnormal tissues (excluding stained tissues) 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 and any other male not siring a pregnancy and testes with gross lesions from 250 and 500 mg/kg bw/day animals were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Postmortem examinations (offspring):

Surviving offspring were terminated via carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were killed by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation.

Examination of offspring was restricted to a macroscopic external examination; except where abnormalities were observed or the offspring died during the study an additional internal examination was performed.

Thyroid Hormone Assessment
Where possible, blood samples taken to produce serum were allowed to clot, centrifuged and the serum from each blood sample was stored frozen at lower than -60 °C for possible evaluation of thyroid hormone. Blood samples to produce plasma were collected into K2EDTA, centrifuged, and the plasma from each blood sample stored frozen at lower than -60 ºC for possible evaluation of thyroid hormones. Samples were taken as follows:
Where possible from each litter, serum samples from two randomly allocated offspring on Day 4 post partum (if offspring were of the same sex, samples from the same litter were pooled). If eight or fewer offspring were present in a litter, then no offspring from that litter were sampled on Day 4 post partum.
Where possible from each litter, serum samples were taken from two randomly allocated offspring (one male and one female) on Day 13 post partum. Where possible from each litter, plasma samples were also taken from two randomly allocated offspring (one male and one female) on Day 13 post partum.

All serum samples were shipped to the test site (Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS) for serum analysis, frozen, packed in dry ice. The serum from adult males and Day 13 offspring were analyzed for Thyroxine (T4) under the supervision of the Prinicpal Investigator (H Bose).

Histopathology
Where possible on Day 13 of age, for one male and one female offspring per litter, the Thyroid/Parathyroids were retained in buffered 10% formalin.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05.

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module.

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

Reproductive indices:

Mating Performance and Fertility
Mating index (%) = (Number of animals mating / Animals paired) x 100

Pregnancy index (%) = (Number of animals achieving pregnancy / Animals mated) x 100


Parturition index (%) = (Number of live litters born / Number pregnant) x 100

Offspring viability indices:

Survival Indices
The following were calculated for each litter:
Post-implantation loss (%) = ((Number of implantation sites - Total number of offspring born) / Number of implantation sites) x 100

Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100

Viability index 1 (%) = (Number of live offspring on Day 4 (before blood sampling) / Number live offspring on Day 1 after littering) x 100

Viability index 2 (%) = (Number of live offspring on Day 13 / Number of live offspring on Day 4) x 100
Viability index 2 takes into consideration the offspring used for blood sampling on Day 4 post partum.


Sex Ratio
Sex ratio was calculated for each litter value on Days 1, 4 and 13 post partum, using the following formula:

(Number of male offspring / Total number of offspring) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs observed for either sex that were considered to indicate any systemic effect of treatment.
All treated animals showed orange fur staining consistent with the colored nature of the test item during the study.
Isolated instances of noisy respiration were apparent for two control males, two females at 500 mg/kg bw/day and one male and one female at 1000 mg/kg bw/day. Additionally, isolated instances of increased post-dosing salivation were apparent for one female at 500 mg/kg bw/day and two males at 1000 mg/kg bw/day. These findings were considered most likely to be related to difficulties in dosing particular animals on these occasions and were considered not to reflect test item toxicity.
One female at 500 mg/kg bw/day showed labored respiration, piloerection and chromodacryorrhea on Day 39, with the chromodacryorrhea persisting until Day 46. The onset of these signs was around the time of expected parturition and was considered to reflect stress/difficulties related to the delivery of the litter for this animal, which showed total litter loss post partum. As no similar instances were apparent for other treated females, this finding was considered to be incidental and unrelated to treatment.
One male receiving 250 mg/kg bw/day showed lethargy on Day 42 of the study. Another male at this dosage showed piloerection on Days 24-27 and piloerection and hunched posture on Day 43. One female at 500 mg/kg bw/day showed generalized fur loss from Day 36 of the study. In the absence of any similar findings at the high dosage of 1000 mg/kg bw/day, these findings were considered incidental and unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of treatment on body weight and body weight gain of males throughout the study at 250, 500 or 1000 mg/kg bw/day.
There was no obvious effect of treatment on body weight and body weight gain of females during the pre-pairing, gestation or lactation phases of the study at 250, 500 or 1000 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of treatment on food consumption of males throughout the pre-pairing and post-pairing phases of the study at 250, 500 or 1000 mg/kg bw/day.
There was no effect of treatment on food consumption of females during the pre-pairing, or gestation phase of the study at 250, 500 or 1000 mg/kg bw/day.
At 1000 mg/kg bw/day, food intake for females was statistically significantly lower than control during days 4-7 and 7-14 of lactation. This lower intake may reflect lower demand from the smaller litter, compared to control, at this dosage. There was no effect of treatment on food consumption of females during the lactation phase of the study at 250 or 500 mg/kg bw/day.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no effect of treatment on food conversion efficiency of either sex during the pre-pairing phase of the study or for males during the post pairing phase of the study at 250, 500 or 1000 mg/kg bw/day.

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

no effects observed

Description (incidence and severity):

Visual inspection of water bottles throughout the study did not indicate any effect of treatment for either sex throughout the study.

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathological examination of reproductive tissues from the control and 1000 mg/kg bw/day animals did not reveal any findings considered to be related to treatment with the test item. In particular there were no consistent treatment-related pathologic findings in the testes following the qualitative examination of the stages of spermatogenesis in the testes (no treatment-related related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or in the ovaries following the evaluation of the follicles and corpora lutea.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Evaluation of Thyroxine (T4) in adult males did not identify any obvious effect of treatment or indication of endocrine disruption at 250, 500 or 1000 mg/kg bw/day.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Assessment of estrous cycles during the pre-pairing phase of the study did not indicate any obvious effect of treatment at 250, 500 or 1000 mg/kg bw/day.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating performance as assessed by the number of paired animals that mated and pre-coital interval was unaffected by treatment at 250, 500 or 1000 mg/kg bw/day.
At 500 mg/kg bw/day, the distribution of pre-coital intervals attained statistical significance when compared to control, but as all but one of these animals mated within the first four days of pairing (i.e. at the first expected estrus opportunity) then this was clearly incidental and unrelated to treatment.
There was no obvious effect on fertility, as assessed by the number of females that achieved pregnancy, at 250, 500 or 1000 mg/kg bw/day.
The intergroup distribution of gestation lengths observed during the study did not indicate any obvious effect of treatment at 250, 500 or 1000 mg/kg bw/day.
One female at 500 mg/kg bw/day, which showed total litter loss post partum, was noted to have a short gestation period, however this isolated finding, in the absence of any similar finding at 1000 mg/kg bw/day, was considered to be incidental and unrelated to treatment.
At 250, 500 and 1000 mg/kg bw/day, there was one non-pregnant female at each dosage, additionally one control female and one female at 500 mg/kg bw/day showed total litter loss post partum.

At 1000 mg/kg bw/day the mean number of implantations was lower than control although differences did not attain statistical significance and the mean value was within the historical control range. To a certain extent, mean values were adversely influenced by one litter (No. 94) that showed unilateral pregnancy, with only two implantations sites being visible in the right horn, however, when this animal was excluded, the mean number of implantation sites still remained lower than control. Mean post-implantation loss was slightly higher than control, but when the female with unilateral pregnancy was removed (this female lost one of her two implantations), the mean value was similar to control.
There was considered to be no effect of maternal treatment on the numbers of implantations, post-implantation loss, litter size at birth/Day 1 and subsequent offspring survival to Day 13 of age at 250 or 500 mg/kg bw/day.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

food efficiency

water consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: systemic toxicity

2

Dose descriptor:

NOEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: fertility

3

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Remarks on result:

other: fertility, growth, development and survival of the offspring

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs apparent for the offspring during the study were generally typical of the age observed and the distribution and low incidence did not indicate any obvious systemic effect of maternal treatment. At all dosages, the majority of offspring showed orange staining of the fur during the second week of lactation consistent with the nature of the test item and indicating at least some external exposure to the test item.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was considered to be no effect of maternal treatment on litter size at birth/Day 1 and subsequent offspring survival to Day 13 of age at 250 or 500 mg/kg bw/day.
Litter size at Day 1 was lower than control due to the previous lower number of implantations and remained lower than control until termination of the litter on Days 13 of age. The lower litter size attained statistical significance from Day 4 of age following the removal of offspring for Day 4 blood sampling.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no obvious effect of maternal treatment on offspring body weight on Day 1 and subsequent body weight gain to Day 13 at 250, 500 or 1000 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any obvious systemic effect of treatment on offspring development at 250, 500 or 1000 mg/kg bw/day.
At all dosages, orange staining of the fur and orange contents within the stomach and gastro-intestinal tract were apparent at necropsy on Day 13 of age. This finding indicates internal and external exposure to the test item and is suggestive of transfer of the test item in the mother’s milk.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of maternal treatment on sex ratio of the litters throughout, indicating that the differences in litter sizes was not associated with any selective effect on survival for either sex.
Sex ratio for the offspring was similar to control and did not indicate any selective effect of maternal treatment on survival for either sex at any of the dosages investigated.
Evaluation of ano-genital distance for female offspring on Day 1 post partum revealed statistically significant longer ano-genital distance, compared to control for all dosages, although group mean values showed no consistent dosage relationship. When normalized for body weight, ano-genital distance remained greater than control but there was still no consistent dosage relationship. All individual litter values normalised for body weight for treated animals were within the historical control range whilst the value for one control litter was below this historical range. For male offspring, ano-genital distance normalized for body weight was also statistically significantly longer than control at 1000 mg/kg bw/day. All individual litter values normalised for body weight for these treated animals were within the historical control range whilst values for two control litters were below the historical range.
Evaluation of visible nipple count for male offspring on Day 13 post partum did not reveal any effect of maternal treatment at 250, 500 or 1000 mg/kg bw/day.
Evaluation of Thyroxine (T4) in offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 250, 500 or 1000 mg/kg bw/day.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

gross pathology

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

not specified

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the No Observed Adverse Effect Level for the adult animals was considered to be 1000 mg/kg bw/day (the highest dosage tested). The No Observed Effect Level for fertility was considered to be 500 mg/kg bw/day. For females at 1000 mg/kg bw/day, there was a non-statistically significant lower mean implantation count; this lower mean value was within the historical control range and was of equivocal relationship to treatment. The No Observed Adverse Effect Level for the growth, development and survival of the offspring 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, to evaluate some endocrine disruptor relevant endpoints 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 29 July 2016).

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 approximately six weeks (males) and eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 250, 500 and 1000 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 13 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 ano-genital distance and visible nipple count (male offspring only).

Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14post partum, respectively. Any female which did not produce a pregnancy was terminated around the same time as littering females. 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 during the study.

Clinical Observations

All treated animals showed orange fur staining consistent with the colored nature of the test item but there were no clinical signs observed for either sex that were considered to indicate any systemic effect of treatment at 250, 500 and 1000 mg/kg bw/day.

Body Weight

Body weight and body weight gain for males throughout the study or for females during the pre-pairing, gestation or lactation phases of the study was unaffected by treatment at 250, 500 or 1000 mg/kg bw/day.

Food Consumption

Food consumption for males throughout the study and for females during the pre-pairing and gestation phases of the study was unaffected by treatment at 250, 500 or 1000 mg/kg bw/day.

At 1000 mg/kg bw/day, food intake for females was statistically significantly lower than control from Day 4 of lactation but was probably attributable to lower litter size at this dosage, compared to control. Food consumption during lactation at 250 or 500 mg/kg bw/day was similar to control.

Food Conversion Efficiency

Food conversion efficiency for either sex during the pre-pairing phase or for males during the post pairing phase of the study was unaffected by treatment at 250, 500 or 1000 mg/kg bw/day.

Water Consumption

Visual inspection of water bottles throughout the study did not indicate any effect of treatment for either sex throughout the study.

Reproductive Performance

Estrous Cycle

Pre-pairing estrous cycles were unaffected by treatment at 250, 500 or 1000 mg/kg bw/day.

Mating

Mating performance was unaffected by treatment at 250, 500 or 1000 mg/kg bw/day.

Fertility

Fertility, as assessed by the number of females that achieved pregnancy, was unaffected by treatment at 250, 500 or 1000 mg/kg bw/day.

Gestation Length

Gestation lengths were unaffected by treatment at 250, 500 or 1000 mg/kg bw/day.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

At 1000 mg/kg bw/day the mean number of implantations was lower than control which resulted in lower litter size from birth until termination (Day 13 of age) although there was no effects of treatment on post-natal offspring survival at this dosage. Sex ratio was unaffected and differences in implantation/litter sizes did not represent any selective effect on survival for either sex.     

There was considered to be no effect of maternal treatment on the numbers of implantations, post-implantation loss, litter size and sex ratio at birth/Day 1 and subsequent offspring survival to Day 13 of age at 250 or 500 mg/kg bw/day.

Offspring Growth and Development

There was no obvious effect of maternal treatment on offspring body weight on Day 1 and subsequent body weight gain to Day 13 at 250, 500 or 1000 mg/kg bw/day. 

Ano-genital distance for female offspring on Day 1post partumwas statistically significantly longer than control, including when normalized for body weight at all dosages, but differences showed no consistent dosage relationship. For male offspring, ano-genital distance normalized for body weight was also statistically significantly longer than control at 1000 mg/kg bw/day.

Visible nipple count for male offspring on Day 13post partumwas unaffected by maternal treatment at 250, 500 or 1000 mg/kg bw/day.

At all dosage, the majority of offspring showed orange staining of the fur during the second week of lactation consistent with the nature of the test item and indicating at least some external exposure to the test item.

Pathology

Necropsy

Offspring

At all dosages, orange staining of the fur and orange contents within the stomach and gastro-intestinal tract were apparent at necropsy on Day 13 of age, indicating internal and external exposure to the test item.    

Adults

Yellow discoloration of the mammary glands and adipose tissue was apparent for the majority of males and females at 250, 500 and 1000 mg/kg bw/day. Additionally yellow/orange discoloration and/or contents were apparent, at a lesser incidence, for the stomach and caecum. These finding are consistent with the nature of the Test Item.

At 1000 mg/kg bw/day, four males showed a mottled appearance to the kidneys, three of these animals also exhibited a mottled appearance to the liver.

Organ Weights

Male reproductive organ weights were unaffected by treatment at 250, 500 or 1000 mg/kg bw/day.

Thyroid weights for either sex were unaffected by treatment at 250, 500 and 1000 mg/kg bw/day.

Histopathology

Histopathological examination of reproductive tissues from the control and 1000 mg/kg bw/day animals did not reveal any findings considered to be related to treatment with the test item. 

Thyroid Hormone Analysis

Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 250, 500 or 1000 mg/kg bw/day.

Conclusion

Based on the results of this study, the No Observed Adverse Effect Level for the adult animals was considered to be 1000 mg/kg bw/day (the highest dosage tested). The No Observed Effect Level for fertility was considered to be 500 mg/kg bw/day. For females at 1000 mg/kg bw/day, there was a non-statistically significant lower mean implantation count; this lower mean value was within the historical control range and was of equivocal relationship to treatment. The No Observed Adverse Effect Level for the growth, development and survival of the offspring was considered to be 1000 mg/kg bw/day.