1-hydroxy-4-(p-toluidino)anthraquinone

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an OECD guideline 421 (Reproduction/Developmental Toxicity Screening Test) Macrolex Violett B was administered by gavage to three groups, each of twelve male and twelve female Wistar rats, for approximately 6 weeks (males) and up to 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) over the same period.

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 No Observed Adverse Effect Level (NOAEL) for the adult animals was considered to be 1000 mg/kg bw/day. 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 (the highest dosage tested).

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 April 2017 Experimental Completion Date: 31 August 2017

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)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Physical State/Appearance: Dark blue/violet powder
Date Received: 10 March 2016
Storage Conditions: Ambient temperature in the dark (formulated in the light)
Expiry Date: 02 June 2020

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 eighteen 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 279 to 346g, and were approximately eleven weeks old. The females weighed 199 to 236g, 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. 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%. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study.

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 as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services as part of Envigo study number XD69DQ and confirmed that test item formulations were stable for at least 16 days when stored at approximately 4 ºC in the dark. Formulations were therefore prepared fortnightly and stored at approximately 4 °C in the dark and used within the known stability period.

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.

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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test item formulations were taken on three occasions and analyzed for concentration of Macrolex Violett B at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 92-101% 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 5 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 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. As staining of the adipose tissue was observed in offspring when thyroids were removed, examination was extended to include an internal examination of two randomly selected offspring. Abnormalities were retained in 10% Buffered 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.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 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:

The dose levels were chosen in collaboration with the Sponsor based on the results of previous toxicity work including a Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat (Envigo Study Number XD69DQ) and a Ninety Day Repeated Dose Oral (Gavage) Toxicity Study in the Rat (Envigo Study Number YL22VX).
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:

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 recorded 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 (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.

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):

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.

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):

Necropsy
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 and offspring, 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 (H Bose). All plasma samples were retained at the Test Facility.

Organ Weights
Thyroid/parathyroid were dissected free from fat for terminal kill animals from both sexes, and weighed post-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-bulbcavernous) muscle
Thyroid/parathyroid
Ovaries
Uterus/Cervix (with oviducts)
Mammary gland
Vagina
Pituitary

♦ preserved in Modified Davidsons fluid

Where possible on Day 13 of age, for one male and one female offspring per litter, theThyroid/Parathyroids were retained in 10% Buffered Formalin.
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: J Schofield). The tissues (excluding adipose tissue) from control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, 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.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell or stage-specificity of testicular findings was noted.

Postmortem examinations (offspring):

Surviving offspring were terminated via 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 initially restricted to a macroscopic external examination, however as staining of the adipose tissue was observed during the removal of thyroids, examination was extended to include an internal examination of two randomly selected offspring. Abnormalities were retained in 10% Buffered 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. 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.

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. Statistical analysis was performed on the following parameters:
Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Implantation Sites, Post-implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Developmental Parameters, Absolute Organ Weights, Body Weight-Relative Organ Weights and Thyroid Hormone (T4).
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
The following parameters were calculated from the individual data during the mating period of the parental generation:
i. Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii. Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals mated / Number of animals paired) x 100

Pregnancy Index (%) = (Number of pregnant females / Number of animals mated) x 100

Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii. Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of females delivering live offspring / Number of pregnant females) x 100

Offspring viability indices:

The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 13 of age).

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

Post–implantation loss (%) = ((Number of implantation sites - Total number of offspring born) / Number of implantation sites) x100

ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1 / Number of offspring born) x 100

Viability Index 1 (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1) x 100

Viability Index 2 (%) = (Number of offspring alive on Day 13 / Number of offspring alive on Day 4) x 100

Viability Index 2 takes into consideration the Offspring used for blood sampling on Day 4 post partum.

iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1, 4 and 13 post partum, using the following formula:
(Number of males offspring / Total number of offspring) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs observed for surviving animals that indicated any obvious systemic effect of treatment for either sex at 250, 500 or 1000 mg/kg bw/day.
Staining of the fur was observed for five males and seven females at 250 mg/kg bw/day, the majority of males and all females at 500 mg/kg bw/day from Day 8 and all animals at 1000 mg/kg bw/day. Additionally staining of the cage bedding and dark faeces were apparent for cages holding 1000 mg/kg bw/day animals at stages throughout the study. These findings were considered to be consistent with the coloured nature of the test item and were considered to be of no toxicological significance.
Noisy respiration was observed for one male at 500 mg/kg bw/day on Day 31 of the study. Increased post-dosing salivation was observed for one male at 500 mg/kg bw/day on Day 34 of the study and for one female at 1000 mg/kg bw/day on Day 35 of the study. The distribution and incidence of these findings indicate that they were most probably associated with occasional difficulties in dosing these particular animals rather than any indication of test item toxicity.

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:

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 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.
For females at 1000 kg bw/day, slightly lower body weight gain was apparent during the first week of treatment compared to control, however, differences did not attain statistical significance and in isolation, this finding was considered to reflect normal biological variation rather than indicating any effect of treatment.

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 250, 500 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 250, 500 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 for 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 at 250, 500 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 examined

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 (testes, epididymides and ovaries) 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.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

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.

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.
No consistent treatment-related pathologic findings in the ovaries following the evaluation of the follicles and corpora lutea.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

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).

Reproductive performance:

no effects observed

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.
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.
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.

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

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

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 performance

Remarks on result:

other: reproductive 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.

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 the numbers of implantations, post-implantation loss, litter size at birth/Day 1 and subsequent offspring survival to Day 13 of age at 250, 500 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 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 effect of maternal treatment at 250, 500 or 1000 mg/kg bw/day.
At all dosages, blue staining of the adipose tissue was apparent at necropsy of offspring at 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):

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 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.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

There were 0, 1, 1 and 0 females that failed to achieve pregnancy in the control, 250, 500 and 1000 mg/kg bw/day dosage groups respectively. The following results are based on the 12, 11, 11 and 12 females successfully rearing young to Day 13 of age at 0 (Control), 250, 500 and 1000 mg/kg bw/day respectively.

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 (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 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 6 weeks (males) and up to 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) over the same 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 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 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 adult 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. 

Clinical Observations

There were no clinical signs observed for surviving animals that indicated any obvious systemic effect of treatment for either sex at 250, 500 or 1000 mg/kg bw/day.

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 dosage of 100, 300 or 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 or for females during the pre-pairing, gestation or lactation phases of the study were unaffected by treatment at 250, 500 or 1000 mg/kg bw/day.

Food Conversion Efficiency

There was no effect of treatment on food conversion efficiency for 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

Visual inspection of water bottles throughout the study did not indicate any effect of treatment for either sex at 250, 500 or 1000 mg/kg bw/day throughout the study

Reproductive Performance

Estrous Cycle

Estrous cycles during the pre-pairing phase of the study 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 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

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, 500 or 1000 mg/kg bw/day. 

Offspring Growth and Development

There was no 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 on Day 1, male visible nipple count on Day 13post partumand clinical signs from birth to termination on Day 13 did not indicate any effect of maternal treatment on the offspring at 250, 500 or 1000 mg/kg bw/day.

Pathology

Necropsy

Offspring

At all dosages, blue staining of the adipose tissue was apparent at necropsy of offspring at 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

Blue discoloration of the mammary glands and adipose tissue was apparent for both sexes at 250, 500 or 1000 mg/kg bw/day. Blue coloured contents were apparent in the stomach and/or cecum for a few animals at all dosages. These findings are consistent with the nature of the test item and were considered unlikely to indicate a systemic effect of treatment 250, 500 or 1000 mg/kg bw/day.

Organ Weights

Thyroid weights for both sexes and male reproductive organ weights appeared unaffected by treatment at 250, 500 or 1000 mg/kg bw/day.  

Histopathology

Histopathological examination of reproductive tissues (testes, epididymides and ovaries) 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 (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

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

Justification for classification or non-classification

In an OECD guideline 421 (Reproduction/Developmental Toxicity Screening Test) the No Observed Adverse Effect Level (NOAEL) for the adult animals was considered to be 1000 mg/kg bw/day. 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 (the highest dosage tested).

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is therefore not justified.