Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a study according to OECD TG 421 (Oral (Gavage) Reproduction/Developmental Toxicity Screening Test in the Rat) Macrolex Rotviolet R was administered by gavage to three treatment groups for approximately six weeks for males and up to eight weeks throughout a two week pre-pairing phase, pairing, gestation and lactation to Day 13 for females at dose levels of 100, 300 and 1000 mg/kg bw/day. 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 Adverse Effect Level for reproduction and for the growth, development and survival of the offspring was also considered 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:
Experimental Starting Date: 29 March 2017 Experimental Completion Date: (date of final thyroid hormone assessment phase report)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Identification: Macrolex Rotviolett R
Physical State/Appearance: Violet solid
Date Received: 07 October 2016
Storage Conditions: Ambient temperature (approximately 10 to 30°C) in the dark, used/formulated in the light
Expiry Date: 28 July 2017
No correction for purity was made.
Species:
rat
Strain:
other: Wistar Han™:RccHan™:WIST
Sex:
male/female
Details on test animals 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 258 to 360g and were approximately eleven weeks old. The females weighed 193 to 238g and were approximately fourteen weeks old. An additional female was subsequently allocated to the 1000 mg/kg/bw/day dosage group due to a non-treatment related death early in the study.

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. Certificates of analysis of the batches of diet used are given in Annex 6. 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. 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.
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 vehicle had previously been successfully used in other toxicity studies. On each day of formulation preparation, for each concentration the required amount of test item was weighed out and added to the required volume of vehicle and shaken/mixed to give a homogeneous bulk formulation. These bulk formulations were subsequently divided into the required daily aliquots and stored at approximately 4°C, in the dark, until the day of use.



.
Details on mating procedure:
On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days. The additional animal at 1000 mg/kg bw/day was paired on day 15 relative to its later start of treatment.
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:
The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK Analytical Services as part of another study (Envigo Study Number: LY77PH), where formulations were shown to be stable for at least eleven days when stored at approximately 4°C, in the dark. Formulations for this study were made and used within the known stability period.

Samples of the test item formulations were taken on three occasions and analyzed for concentration of Macrolex Rotviolett R at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 91-103% of the nominal concentration.
Duration of treatment / exposure:
approximately six weeks for males and up to eight weeks throughout a two week pre-pairing phase, pairing, gestation and lactation to Day 13 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). At 1000 mg/kg bw/day, an additional female was subsequently allocated to the study on Day 3 due to a non-treatment related death early in the study. 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. The additional animal at 1000 mg/kg bw/day was paired on day 15 relative to its later start of treatment.
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, 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.
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. As staining of the adipose tissue was observed for some offspring, examination was extended to exclude an additional internal examination on two randomly selected male and two female offspring for each litter. Abnormal tissues were retained in buffered 10% formalin.
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. Blood samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 males and 12 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose levels were selected in consultation with the Study Monitor and based on available toxicity data including a Twenty-Eight Day Repeated Dose Oral (Gavage) Toxicity Study in the rat (Envigo Study Number JQ66PW) In the twenty-eight day toxicity study, a high dosage of 1000 mg/kg bw/day appeared to be well tolerated and therefore this dosage was selected as the high dosage for this study, with lower dosages of 100 and 300 mg/kg bw/day also being utilized.
# On Day 2 relative to the start of dosing (18 April 2017), Female 96 treated with 1000 mg/kg bw/day was terminated due to the presence of a mass in the ano-genital region. As this death was considered unrelated to treatment with the test item, another female (from the spare animals on this study) was included into this dosage group from 19 April 2017 (Day 3) in order to maximize the reproductive assessment; all procedures for the newly added female were performed relative to its dose start date of 19 April 2017.
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 4 mL/kg of Arachis oil BP.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
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. Bodyweights were also recorded prior to terminal kill.

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 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 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 13 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. 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 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.

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 (A Peard).

Organ Weights
The epididymides, testes, seminal vesicles (with coagulating 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 partial fixation.

3.5.2 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

Additionally, due to staining observed at necropsy, adipose tissue was retained for all adult animals at termination.
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 tissues (excluding adipose tissue) from control and 1000 mg/kg bw/day dose group animals, and any animals which failed to mate or 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. The animal at 1000 mg/kg bw/day that was terminated early was not subjected to microscopic examination as this death was clearly unreleated to treatment.



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.

Initially examination of offspring was restricted to a macroscopic external examination, except where abnormalities were observed an additional internal examination was performed. As staining of the adipose tissue was observed for some offspring, examination was extended to include an additional internal examination on two randomly selected male and two female offspring for each litter. Abnormal tissues were retained in buffered 10% formalin.

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.

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 (A Peard).

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

Fertility index (%) = (Number of animals achieving pregnancy / Animals pairing) 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


Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no clinical signs observed that indicated any systemic effect of treatment at dosage of 100, 300 or 1000 mg/kg bw/day.
At 1000 mg/kg bw/day, fur staining was apparent for all animals at some stage of the study, being first observed for males on Day 4 of the study and for females on Day 8. Fur staining was also apparent for the majority of males at 300 mg/kg bw/day, being first observed on Day 11 of the study. For females at this dosage, fur staining was limited to one female on two occasions. At 100 mg/kg bw/day, fur staining was restricted to two males on three occasions during the study. Additionally, three males at 300 mg/kg bw/day and two males at 1000 mg/kg bw/day showed staining around the snout during the study. One male at 100 mg/kg bw/day showed staining of the ano-genital region. These clinical signs were considered to be consistent with the coloured nature of the test item.
Increased post dosing salivation was observed for two males at 300 mg/kg bw/day and five males at 1000 mg/kg bw/day on occasions during the study. One male at 100 mg/kg bw/day showed noisy respiration on Day 14 of the study. These isolated findings were considered most likely to reflect difficulties in the dosing procedure for particular animals and their distribution and frequency did not indicate any toxicological effect of treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was one unscheduled death on the study. At 1000 mg/kg bw/day, female 96 was observed to have a mass (approximately 8mm x 8mm) in the ano-genital area on Day 2 and was killed for animal welfare considerations. This death, in the early stages of the study, was clearly unrelated to treatment, and therefore an additional female (number 97) was added to study to bring the groups complement of animals back up to twelve.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect treatment on body weight gain for males throughout the study at 100, 300 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 100, 300 or 1000 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
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 100, 300 or 1000 mg/kg bw/day.
There was no effect of treatment on food consumption of females during the pre-pairing, gestation or lactation phases of the study at 100, 300 or 1000 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 100, 300 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 at 100, 300 or 1000 mg/kg bw/day.
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
Description (incidence and severity):
There were no statistically significant differences from control for thyroid weights of either sex at 100, 300 or 1000 mg/kg bw/day.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Purple discoloration of the mammary glands and/or adipose tissue was apparent for all or the majority of males and females (including the decedent animal) at 100, 300 and 1000 mg/kg bw/day. Additionally purple coloured contents were apparent in the stomach for the majority of males and a few females at 1000 mg/kg bw/day, one male and a few females at 300 mg/kg bw/day and a few males at 100 mg/kg bw/day. These findings are consistent with the nature of the test item and were considered unlikely to indicate a systemic effect of treatment.
Neuropathological findings:
not examined
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 and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 100, 300 or 1000 mg/kg bw/day.
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 100, 300 or 1000 mg/kg bw/day.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
At 300 mg/kg bw/day, two males showed small and flaccid testes and small epididymides, both of these males failed to mate with their female partner. In the absence of any similar finding at the high dosage of 1000 mg/kg bw/day, these findings were considered to be incidental and unrelated to treatment.
There were no statistically significant differences from control for male reproductive organ weights at 100, 300 or 1000 mg/kg bw/day that were considered to be related to treatment. At all dosages, absolute and body weight relative epididymal weights were lower than control, however group mean values showed no dosage relationship. With the exception of two males at 300 mg/kg bw/day, all individual epididymal weights for treated animals were within the historical control range, additionally individual epididymal weights for one control animal exceeded the historical control range.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Mating
Mating performance as assessed by the number of paired animals that mated and pre-coital interval was unaffected by treatment at 100, 300 or 1000 mg/kg bw/day.
At 300 mg/kg bw/day, two pairs of animals failed to mate during the two week pairing period, however in the absence of any similar occurrence at the highest dosage of 1000 mg/kg bw/day, this finding was considered incidental and unrelated to treatment.

Fertility
There was no obvious effect on fertility, as assessed by the number of females that achieved pregnancy, at 100, 300 or 1000 mg/kg bw/day.

Gestation Length
The intergroup distribution of gestation lengths observed during the study did not indicate any obvious effect of treatment at 100, 300 or 1000 mg/kg bw/day.

Three control females and one female at 300 mg/kg bw/day, and one female at 1000 mg/kg bw/day did not achieve pregnancy.
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
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: reproductive performance
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs apparent for the offspring during the study were generally typical of the age observed and neither the distribution nor incidence of these findings indicated any effect of maternal treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
One female at 100 mg/kg bw/day and one female at 300 mg/kg bw/day had total litter losses.
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 100, 300 or 1000 mg/kg bw/day.
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 100, 300 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 maternal treatment at 100, 300 or 1000 mg/kg bw/day.
At all dosages, pink staining of the adipose tissue was apparent at necropsy on Day 13 of age. This finding indicates internal 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:
no effects observed
Description (incidence and severity):
Sex ratio for the offspring was similar to control in all treated groups 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 offspring on Day 1 post partum and visible nipple count for male offspring on Day 13 post partum did not reveal any effect of maternal treatment at 100, 300 or 1000 mg/kg bw/day.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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
Critical effects observed:
no
Reproductive effects observed:
no
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 Adverse Effect Level for reproduction and for the growth, development and survival of the offspring was also 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 treatment groups for approximately six weeks for males and throughout a two week pre-pairing phase, pairing, gestation and lactation to Day 13 for females, at dose levels of 100, 300 and 1000 mg/kg bw/day. Each treatment groups initially consisted of twelve male and twelve female Wistar HanTM:RccHamTM:wist strain rats, however an additional female was allocated to the 1000 mg/kg bw/day doseage group due to a non-treatment related death early in the study. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP) over the same treatment period. 

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 on Day 1 and visible nipple count on Day 13 (male offspring only).

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all non-recovery treated females including controls through pre-pairing, pairing and up to confirmation of mating.

Vaginal smears were also performed in the morning on the day of termination for all non-recovery treated females.

Adult males were terminated on Day 44, followed by the termination of all adult females and offspring on Days 14 and 13 post 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 on the study that were considered to be related to treatment. At 1000 mg/kg bw/day, one female was killed due to the presence of an ano-genital mass on Day 2 but this death in the early stages of the study was clearly unrelated to treatment. 

Clinical Observations

Fur staining, consistent with the colored nature of the test item, was observed for all animals at 1000 mg/kg bw/day and was also apparent, at a lower incidence, at 100 and 300 mg/kg bw/day. However, there were no clinical signs observed during the study that indicated any systemic effect of treatment at dosages of 100, 300 or 1000 mg/kg bw/day.

Body Weight

There was no effect treatment on body weight or body weight gain for males throughout the study or for females during the pre-pairing, gestation or lactation phases of the study at 100, 300 or 1000 mg/kg bw/day.

Food Consumption

Food consumption for males throughout the study and for females during the pre-pairing, gestation or lactation phases of the study was unaffected by treatment at 100, 300 or 1000 mg/kg bw/day.

Food Conversion Efficiency

There was no effect treatment on food conversion efficiency for males throughout the study or for females during the pre-pairing, gestation or lactation phases of the study at 100, 300 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 at 100, 300 or 1000 mg/kg bw/day.

 

 

Reproductive Performance

Estrous Cycle

Pre-pairing estrous cycles were unaffected by treatment at 100, 300 or 1000 mg/kg bw/day.

Mating

Mating performance was unaffected by treatment at 100, 300 or 1000 mg/kg bw/day.

Fertility

Fertility was unaffected by treatment at 100, 300 or 1000 mg/kg bw/day.

Gestation Length

Gestation lengths were unaffected by treatment at 100, 300 or 1000 mg/kg bw/day

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

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 100, 300 or 1000 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 100, 300 or 1000 mg/kg bw/day. 

Ano-genital distance for offspring on Day 1 of age, visible nipple count for male offspring on Day 13 of age and clinical signs apparent for the offspring to Day 13 of age were unaffected by maternal treatment at 100, 300 or 1000 mg/kg bw/day.

Pathology

Necropsy

Offspring

At all dosages, pink staining of the adipose tissue was apparent at necropsy on Day 13 of age. This finding indicates internal exposure to the test item and is suggestive of transfer of the test item in the mother’s milk.

Adults

Purple discoloration of the mammary glands and/or adipose tissue was apparent for the both sexes at 100, 300 and 1000 mg/kg bw/day. Additionally purple coloured contents were apparent in the stomach for animals at 1000 mg/kg bw/day and, to a lesser extent, at 100 and 300 mg/kg bw/day. These findings are consistent with the colored nature of the test item.

Organ Weights

Male reproductive organ weights were unaffected by treatment at 100, 300 or 1000 mg/kg bw/day.

Thyroid weights of either sex were unaffected by treatment at 100, 300 or 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 100, 300 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 Adverse Effect Level for reproduction and for the growth, development and survival of the offspring was also 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:
GLP guideline study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Toxicity to reproduction: other studies

Description of key information

No data.

Justification for classification or non-classification

In an OECD guideline 421study (Reproduction/Developmental Toxicity Screening Test) 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 Adverse Effect Level for reproduction and for the growth, development and survival of the offspring was also considered to be 1000 mg/kg bw/day. According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is therefore not justified.