3-methyl-6-(p-toluidino)-3H-dibenz[f,ij]isoquinoline-2,7-dione

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A OECD TG 421 “Reproduction/Developmental Toxicity Screening Test” study is avaialble.

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar 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 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400) over the same treatment period. The No Observed Adverse Effect Level (NOAEL) 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: 5 July 2017 Experimental Completion Date:

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:

no

Remarks:

No deviations occured that were considered to have affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Test item: Macrolex Rot 5B
Appearance: Orange powder (with a yellow cast).
Storage conditions: Red Solid
Expiry Date: 19 August 2021
Receipt Date: 06 February 2017

Species:

rat

Strain:

Wistar

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 293 to 359g and were approximately eleven weeks old. The females weighed 198 to 239g, and were approximately twelve 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. 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; 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.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
No correction for purity was made.
Method of preparation: For the purpose of this study the test item was prepared at the appropriate concentrations as a suspension in Polyethylene glycol 400. 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 18 days when stored refrigerated in the dark. Formulations were therefore prepared three times during the treatment period and stored at approximately 4 ºC 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 each test item formulation were taken and analyzed for concentration of Macrolex Rot 5B at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 96-107% of the nominal concentration confirming the suitability of the formulation procedure employed on the study.

Duration of treatment / exposure:

Approximately six weeks for males and up to eight weeks for 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. For the 14 days prior to pairing, pre-pairing vaginal smears were performed and assessed for females.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. 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.
v. 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.
vi. 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.
vii. The male dose groups were killed and examined macroscopically on Day 44 or 45.
viii. 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/parathyroid samples were also retained from one male and one female from each litter where litter sizes allowed. All surviving offspring were killed and examined externally. Where possible, an internal necropsy was performed on at least one male and one female offspring per litter, any decedent offspring, and offspring showing external abnormalities.
ix. All females were sacrificed 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 sacrificed and examined macroscopically around the same time as littering females. In addition, blood samples to produce both serum and plasma were taken from all adult 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.:

500 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 test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 6 mL/kg of Polyethylene glycol 400.
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.

The dose levels were chosen based on the results of previous toxicity work (Envigo study MS88CX). 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.

Parental animals: Observations and examinations:

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 and 7 post partum. Body weights were also recorded for all animals at 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 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.

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 estrous 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

Thyroid Hormone Analysis
Blood samples taken to produce serum were allowed to clot, centrifuged and the serum from each blood sample stored frozen at lower than -60ºC. Blood samples taken to produce plasma were collected into K2EDTA, centrifuged, and the plasma from each blood sample stored frozen at lower than -60ºC. Samples were taken as follows:
Serum and plasma samples were taken from all adult males and females at termination.
All serum samples were dispatched to the Test Site (Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS) where the serum from adult males and Day 13 offspring was analyzed for Thyroxine (T4) under the supervision of the Principal Investigator (H Bose). All plasma samples were retained at the Test Facility.

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:

Litter Data
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.

Thyroid Hormone Analysis
Blood samples taken to produce serum were allowed to clot, centrifuged and the serum from each blood sample stored frozen at lower than -60ºC. Blood samples taken to produce plasma were collected into K2EDTA, centrifuged, and the plasma from each blood sample stored frozen at lower than -60ºC. Samples were taken as follows:
Where possible serum samples were taken from two randomly allocated offspring from each litter on Day 4 post partum (two females were possible to maximize the number of males for assessment of nipple counts on Day 13 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, serum samples were taken from two randomly allocated offspring per litter (one male and one female) on Day 13 post partum. Where possible, plasma samples were also taken from two randomly allocated offspring per litter (one male and one female) on Day 13 post partum. If required the number/sex of offspring sampled was altered depending on the litter constituents.
All serum samples were dispatched to the Test Site (Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS) where the serum from adult males and Day 13 offspring was analyzed for Thyroxine (T4) under the supervision of the Principal Investigator (H Bose). All plasma samples were retained at the Test Facility.

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 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.

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 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:
Epididymides♦
Prostate
Glans penis
Seminal Vesicles (with coagulating gland)
Gross lesions
Testes♦
LABC (levator ani-bulbocavernous) muscle
Thyroid/Parathyroid
Mammary gland
Uterus/Cervix (with oviducts)
Ovaries
Vagina
Pituitary
♦ preserved in Modified Davidsons fluid


All tissues were dispatched to the histology processing Test Site (Propath UK Ltd.,Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Fower). The tissues from control and 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.

As there were no indications of treatment-related changes, examinations were not extended to include animals in the low and intermediate groups.

Postmortem examinations (offspring):

Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were terminated 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 for one male and one female offspring, any decedent offspring, and any offspring with external abnormalities, where an additional internal necropsy was performed.

Where possible on Day 13 of age, for one male and one female offspring per litter, the thyroid/parathyroids were retained in 10% Buffered 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



Gestation Length and Index
Parturition index (%) = (Number of live litters born / Number pregnant) x 100

Offspring viability indices:

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 (%) = (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 after littering / 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, 7 and 13 post partum, using the following formula:
Percentage males = (Number of males in litter / Total number of offspring in litter) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs observed that indicated any obvious systemic effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.
Staining of the fur was observed for five males and two females from Day 27 and 30 respectively at 100 mg/kg bw/day, all males and seven females from Day 2 and 14 respectively at 300 mg/kg bw/day and all males and females from Day 6 and 5 respectively at 1000 mg/kg bw/day. Additionally dark faeces were apparent for cages holding 300 or 1000 mg/kg bw/day animals throughout most of the study from Day 2. These findings were consistent with the coloured nature of the test item and were considered to be of no toxicological significance.
Noisy respiration was observed for one control male, two males and two females at 100 mg/kg bw/day, two males and four females at 300 mg/kg bw/day and eight males and six females at 1000 mg/kg bw/day. These incidences of noisy respiration were considered to reflect occasional difficulties in dosing isolated animals on particular occasions during the study and also, for treated animals, the high viscosity of the dosing formulations, rather than any indication of a systemic effect of treatment. Isolated incidences of increased post-dosing salivation observed during the study for one female at 100 mg/kg bw/day and three males and seven females at 1000 mg/kg bw/day were also considered to reflect occasional difficulties in dosing isolated animals on occasions during the study.
The incidence of other clinical signs on the study were considered to be incidental and unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths on the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of treatment on body weight and body weight gain of males throughout the study at 100, 300 or 1000 mg/kg bw/day.
Males at 300 or 1000 kg bw/day showed statistically significant higher mean body weight gain during their final week on the study, however values showed no dosage relationship and this finding was considered to reflect normal biological variation rather than any effect of treatment.
There was no 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.
Females at 300 or 1000 kg bw/day showed statistically significant higher mean body weight gain during the first week on the study, but differences showed no dosage relationship. Additionally, at 1000 mg/kg bw/day, higher body weight on Day 14 and 20 of gestation attained statistical significance when compared with control. At 100 mg/kg bw/day, body weight gain during Days 1-4 of lactation was also statistically significantly higher than control. These differences from control for treated animals were considered to reflect normal biological variation rather than any effect of treatment.

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 100, 300 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 100, 300 or 1000 mg/kg bw/day.
Food consumption for treated females was higher than control throughout lactation, however differences did not attain statistical significance and during the second week of lactation showed no dosage relationship. This apparent increase was considered to be incidental and unrelated to treatment.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Intergroup differences in food conversion efficiency did not indicate any effect of treatment on food utilization for 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 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, no consistent treatment-related pathologic findings in the testes following the qualitative examination of the stages of spermatogenesis in the testes (no treatment-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 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:

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 100, 300 or 1000 mg/kg bw/day.
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.
It was noted that two females at 1000 mg/kg bw/day failed to achieve pregnancy, whilst all other control and treated females were pregnant. For one of these non-pregnant females, the male partner showed tubular atrophy in the testes and asperma in the epididymides, however no microscopic change was apparent for the testes or epididymides of the male partnered to the other non-pregnant female. No consistent mechanism or effect of treatment could be attributed to the incidence of non-pregnancy, and whilst this appeared to cluster at the high dosage, the number of females at 1000 mg/kg bw/day was well within the expectations and this finding was considered to be incidental and unrelated to treatment.
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.

There was considered to be no effect of maternal treatment on the numbers of implantations, post-implantation loss, litter size at birth/Day 1

At 1000 mg/kg bw/day, the number of implantations was statistically significantly higher than control, however this was considered to reflect a slightly lower than expected value for the control (two litters with five or less offspring) rather than any effect of treatment on implantation count.

As previously indicated two females failed to achieve pregnancy in the 1000 mg/kg bw/day dosage group; additionally one control female and one female treated at 100 mg/kg bw/day showed total litter loss post partum.

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 toxicity

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

Critical effects observed:

no

Clinical signs:

no effects observed

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 effect of maternal treatment at 100, 300 or 1000 mg/kg bw/day.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

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

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no 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:

no effects observed

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 effect of maternal treatment at 100, 300 or 1000 mg/kg bw/day.

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.

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 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

body weight and weight gain

gross pathology

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) 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, 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 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400) 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 ano-genital distance and visible nipple count (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 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 treated females.

Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14 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. All offspring were examined externally. Where possible, an internal necropsy was performed on at least one male and one female offspring per litter, any decedent offspring, and any offspring showing external abnormalities.

Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis; samples from adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Results…….

Adult Responses

Mortality

There were no unscheduled deaths on the study.

Clinical Observations

There were no clinical signs observed that indicated any obvious systemic effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.

Staining of the fur was apparent for a few animals at 100 mg/kg bw/day, the majority of animals at 300 mg/kg bw/day and all animals at 1000 mg/kg bw/day. Additionally dark faeces were also apparent at 300 and 1000 mg/kg bw/day throughout most of the study. These findings were consistent with the colored nature of the Test Item. Incidences of noisy respiration were also observed during the study at all dosages and for treated animals this was considered to reflect occasional difficulties in dosing isolated animals and the high viscosity of the dosing formulations.    

Body Weight

There was no effect of treatment on body weight and body weight gain of males throughout the study at 100, 300 or 1000 mg/kg bw/day. 

There was no 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

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, or gestation phase of the study at 100, 300 or 1000 mg/kg bw/day.

Food Conversion Efficiency

There was no effect of treatment on food conversion efficiency for either sex throughout the pre-pairing phase or for males during the post pairing phase of the study at 100, 300 or 1000 mg/kg bw/day. 

Water Consumption

There was no effect of treatment on water consumption for either sex at 100, 300 or 1000 mg/kg bw/day.

Reproductive Performance

Estrous Cycle

There was no effect of treatment on estrous cycles of females 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

There was no effect on fertility at 100, 300 or 1000 mg/kg bw/day.

It was noted that two females at 1000 mg/kg bw/day failed to achieve pregnancy, while all other control and treated females were pregnant. Although the occurrence of non-pregnancy clustered at the high dosage, the number of females at 1000 mg/kg bw/day was well within expectations and this finding was considered to be incidental and unrelated to treatment.   

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.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was 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 effect of maternal treatment on ano-genital distance and offspring body weight on Day 1, subsequent body weight gain to Day 13 and visible nipple count for male offspring on Day 13post partumat 100, 300 or 1000 mg/kg bw/day. Additionally clinical signs of the offspring to Day 13 of age did not indicate any effect of maternal treatment at 100, 300 or 1000 mg/kg bw/day.

Pathology

Necropsy

Offspring

Macroscopic necropsy findings for offspring did not indicate any effect of maternal treatment at 100, 300 or 1000 mg/kg bw/day.

Adults

Macroscopic necropsy findings for adult animals did not indicate any effect of maternal treatment at 100, 300 or 1000 mg/kg bw/day.

Organ Weights

There were no statistically significant differences from control for thyroid weights of either sex or for male reproductive organ weights at 100, 300 or 1000 mg/kg bw/day.

Histopathology

Histopathological examination of reproductive tissues did not indicate any effect of treatment at 1000 mg/kg bw/day.

Thyroid Hormone Analysis

Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any 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 (NOAEL) 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

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

Description of key information

A OECD TG 421 “Reproduction/Developmental Toxicity Screening Test” study is avaialble.

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar 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 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400) over the same treatment period. The No Observed Adverse Effect Level (NOAEL) 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 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

Justification for classification or non-classification

A OECD TG 421 “Reproduction/Developmental Toxicity Screening Test” study is avaialble. The No Observed Adverse Effect Level (NOAEL) 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. No classification is warrated.