Tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 28 June 2017 , Experimental Completion Date: 20 December 2018 , Date of final thyroid hormone analysis phase report: 28 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Remarks:

No deviations that affected the scientific purpose and integrity of the study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]

EC Number:

258-605-2

EC Name:

Tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]

Cas Number:

53523-90-3

Molecular formula:

C30H20Li4N4O12S2

IUPAC Name:

tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]

impurity 1

Reference substance name:

unidentified impurity

Molecular formula:

unknown

IUPAC Name:

unidentified impurity

Test material form:

solid

Specific details on test material used for the study:

Identification: Bayscript Gelbkomponente
Physical State/Appearance: Red/brown powder
Chemical Name: Tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]
Date Received: 07 October 2016
Storage Conditions: Stored at ambient temperature in darkness over silica gel.
Used/formulated in light at ambient humidity.
Expiry Date: 17 June 2018
Dietary concentrations were adjusted for purity of the Test Item.

Test animals

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 277 to 364g and were approximately eleven weeks old. The females weighed 189 to 242g and were approximately fourteen weeks old.

Animal Care and Husbandry
Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
The animals were allowed free access to food and water. A ground or powdered diet (Rodent PMI 5002 (Certified) Ground Diet (BCM IPS Limited, London, 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.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was incorporated into the diet at concentrations of 500, 1000 and 2500 ppm at the designated Test Site (Envigo CRS Limited, Huntingdon) under the supervision of the Principal Investigator (Hayley Walters).
The stability and uniformity of the admixtures was determined by Envigo Research Limited, Shardlow, UK, Analytical Services as part of this study. Results showed the formulations to be stable when stored at ambient temperature for seven weeks. Formulations were prepared and used within this stability period. When in use, aliquots were stored at room temperature in labelled bags in labelled, covered bins.

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 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each test item dietary admixture was sampled on all five occasions (2x 200g) from top, middle and bottom and then analyzed for uniformity of distribution and achieved concentration of Bayscript Gelbkomponente by Envigo Research Ltd, Shardlow, UK Analytical Services. The results indicate that the prepared formulations were within 99-132% 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:

Continuous

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. Animals received treated diet, according to dose group throughout the study period. Control animals received basal laboratory diet. The first day of treatment 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 examination was performed for at least one male and one female offspring per litter.
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 litter 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).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 ppm

Remarks:

Adjusted for test item purity

Doses / concentrations 2

Dose / conc.:

1 000 ppm

Remarks:

Adjusted for test item purity

Doses / concentrations 3

Dose / conc.:

2 500 ppm

Remarks:

Adjusted for test item purity

No. of animals per sex per dose:

12 males and 12 females per dose

Control animals:

yes, plain diet

Details on study design:

The dose levels were chosen in collaboration with the Sponsor and were based on the results of previous toxicity work including a Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity and Fourteen Day Repeated Dose Oral (Dietary) Range-Finding Toxicity/Palatability Study in the Rat (Envigo Study number NR32HS).

Examinations

Parental animals: Observations and examinations:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change once daily. 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, 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. Mean achieved dosages for females during gestation and lactation have been calculated, however, these values may have been affected by offspring growth during gestation and the possibility of offspring starting to eat the diet during the final week of treatment.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.

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):

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 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, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Thyroid Hormone Assessment
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 (I Komjarova).

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. Liver and kidneys were removed from terminal kill animals of both sexes, 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 fixation (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-bulbcavernous) muscle
Thyroid/parathyroid
Liver
Uterus/Cervix (with oviducts)
Kidneys
Vagina
Mammary gland
Ovaries
Pituitary

♦ preserved in Modified Davidsons fluid

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.
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 (excluding the liver and kidneys) from control and 2500 ppm (adjusted for purity) dose group animals, and any females which did not produce a litter were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any tissues showing macroscopic abnormalities were also 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 2500 ppm (adjusted for purity) males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

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 per litter, where possible, where an internal examination was performed.

Thyroid Hormone Assessment
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 (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 (I Komjarova).

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 (Thyroxine).
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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs apparent for animals throughout the study.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during 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 adjusted dietary concentrations of 500, 1000 or 2500 ppm. For males receiving 2500 ppm, lower body weight gain attained statistical significance when compared to control during the second week of treatment but, in isolation, this finding appeared incidental and of no toxicological significance
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 adjusted dietary concentrations of 500, 1000 or 2500 ppm. For females receiving 500 or 1000 ppm, lower cumulative body weight gain between Days 1 and 14 of lactation attained statistical significance when compared to control. However, in the absence of any obvious dietary concentration relationship this finding was considered to be incidental and unrelated to dietary exposure to the Test Item.

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 adjusted dietary concentrations of 500, 1000 or 2500 ppm. There was no effect of treatment on food consumption of females during the pre-pairing, gestation or lactation phases of the study at adjusted dietary concentrations of 500, 1000 or 2500 ppm.

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 adjusted dietary concentrations of 500, 1000 or 2500 ppm.

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 adjusted dietary concentrations of 500, 1000 or 2500 ppm, 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:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathological examination of reproductive tissues from the control and animals receiving an adjusted dietary concentration of 2500 ppm did not reveal any findings considered to be related to dietary exposure to 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 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.
Examination of the livers showing macroscopic findings at necropsy did not reveal any evidence of any histopathological change at adjusted dietary concentrations of 500, 1000 or 2500 ppm.

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 dietary exposure to the Test item or indication of endocrine disruption change at adjusted dietary concentrations of 500, 1000 or 2500 ppm.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Assessment of estrous cycles during the pre-pairing phase of the study did not indicate any effect of treatment at adjusted dietary concentrations of 500, 1000 or 2500 ppm.

Reproductive function: sperm measures:

no effects observed

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 adjusted dietary concentrations of 500, 1000 or 2500 ppm.
There was no effect on fertility, as assessed by the number of females that achieved pregnancy, at adjusted dietary concentrations of 500, 1000 or 2500 ppm.
The intergroup distribution of gestation lengths observed during the study did not indicate any obvious effect of dietary exposure at adjusted dietary concentrations of 500, 1000 or 2500 ppm.
All females on the study achieved pregnancy, however one female at an adjusted dietary concentration of 1000 ppm had only a single implantation site in the uterus at necropsy and was not observed to have given birth to a litter. In isolation this finding was considered to be unrelated to dietary exposure to the test item.
There was considered to be no effect of test item exposure on the numbers of implantations, post-implantation loss, litter size at adjusted dietary concentrations of 500, 1000 or 2500 ppm.

Details on results (P0)

Achieved Dosage
Achieved intakes for both sexes within each dietary concentration were considered to generally reflect the selected intervals between the dietary concentration (i.e. the two and five fold increase from the lowest dietary level). Achieved concentration were similar for both sexes within each dietary concentration during the pre-mating phase. As anticipated, achieved intakes, in comparison to males, generally increased for females during late gestation and during lactation reflecting the increasing physiological demand on the females from the litter during these study phases.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

food efficiency

water consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: systemic toxicity

2

Dose descriptor:

NOAEL

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: reproductive toxicity

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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 incidence did not indicate any obvious effect of test item exposure at adjusted dietary concentrations of 500, 1000 or 2500 ppm.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was considered to be no effect of test item exposure on litter size at birth/Day 1 and subsequent offspring survival to Day 13 of age at adjusted dietary concentrations of 500, 1000 or 2500 ppm.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of test item exposure on offspring body weight on Day 1 at adjusted dietary concentrations of 500, 1000 or 2500 ppm.
At 2500 ppm, body weight gain of the offspring was slightly lower than control from Day 4, with differences attaining statistical significance for both sexes during Days 4-7, and for female offspring during Days 7-13; in addition, cumulative body weight gain of the female offspring from Day 1 was statistically significantly lower than control for Days 7 and 13. Despite these observed differences in offspring body weight gain, there were no statistically significant differences from control for offspring body weight and litter weights throughout the study.
Offspring body weight, offspring body weight gain and litters weights appeared unaffected by exposure to the test item at adjusted dietary concentrations of 500 or 1000 ppm.

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 effect of test item exposure at adjusted dietary concentrations of 500, 1000 or 2500 ppm.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio for the offspring was similar to control at all adjusted dietary concentrations and did not indicate any selective effect on survival for either sex at any of the adjusted dietary concentrations 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 test item exposure at adjusted dietary concentrations of 500, 1000 or 2500 ppm.
Evaluation of Thyroxine (T4) in offspring at Day 13 of age did not identify any obvious effect of dietary exposure to the Test item or indication of endocrine disruption change at adjusted dietary concentrations of 500, 1000 or 2500 ppm.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

gross pathology

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for the adult animals was considered to be the adjusted dietary concentration of 2500 ppm (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 the adjusted dietary concentration of 2500 ppm.

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 dietary admixture to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks for males and eight weeks for females (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at adjusted dietary concentrations of 500, 1000 and 2500 ppm. This was equivalent to a mean achieved dosage of 33, 77 and 177 mg/kg bw/day for males respectively and for females 48, 111 and 255 mg/kg bw/day during maturation, 48, 123 and 261 mg/kg bw/day during gestation and 93, 224 and 489 mg/kg bw/day during lactation respectively (incorporating a correction factor for 86.3% purity). A control group of twelve males and twelve females were treated with basal laboratory diet 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 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 14post partum, respectively. Any female which did not produce a litter 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 examination was performed for at least one male and one female offspring per litter. 

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 13post 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 during the study.

Clinical Observations

There were no clinical signs apparent for animals throughout the study.

Body Weight

There was no effect of treatment on body weight and body weight gain of males throughout the study or females during the pre-pairing, gestation or lactation phases of the study at adjusted dietary concentrations of 500, 1000 or 2500 ppm.

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 adjusted dietary concentrations of 500, 1000 or 2500 ppm. 

Food Conversion Efficiency

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 adjusted dietary concentrations of 500, 1000 or 2500 ppm. 

Water Consumption

There was no effect of treatment on water consumption for either sex at adjusted dietary concentrations of 500, 1000 or 2500 ppm. 

Reproductive Performance

Estrous Cycle

There was no effect of treatment on estrous cycles at adjusted dietary concentrations of 500, 1000 or 2500 ppm. 

Mating

There was no effect of treatment on mating performance at adjusted dietary concentrations of 500, 1000 or 2500 ppm. 

Fertility

There was no effect of treatment on fertility at adjusted dietary concentrations of 500, 1000 or 2500 ppm. 

Gestation Length

There was no effect of treatment on gestation lengths at adjusted dietary concentrations of 500, 1000 or 2500 ppm. 

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no effect of treatment on litter size, offspring viability or offspring sex ratios at adjusted dietary concentrations of 500, 1000 or 2500 ppm. 

Offspring Growth and Development

There was no effect of test item exposure on offspring body weight on Day 1 at adjusted dietary concentrations of 500, 1000 or 2500 ppm. 

At 2500 ppm, body weight gain of the offspring was slightly lower than control from Day 4, but offspring body weight and litter weights remained comparable to controls throughout the study. Offspring body weight, offspring body weight gain and litters weights appeared unaffected by exposure to the test item at adjusted dietary concentrations of 500 or 1000 ppm.   

Evaluation of ano-genital distance for offspring on Day 1post partumand visible nipple count for male offspring on Day 13post partumdid not reveal any effect of test item exposure at adjusted dietary concentrations of 500, 1000 or 2500 ppm.

Offspring Observations

There was no obvious effect of test item exposure on the clinical signs apparent for offspring at adjusted dietary concentrations of 500, 1000 or 2500 ppm. 

Pathology

Necropsy

Offspring

There was no effect of test item exposure on the macroscopic necropsy findings for offspring at adjusted dietary concentrations of 500, 1000 or 2500 ppm.

Adults

There were a number of liver findings observed on the study, including enlarged, pale/pale area, or dark coloration at adjusted dietary concentrations of 2500 ppm and 500 ppm. A female receiving an adjusted dietary concentration of 1000 ppm showed a mass on the liver. These findings were not associated with any histopathological change and therefore were considered to be of no toxicological significance.

Thyroid Hormone Analysis

Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of dietary exposure to the Test item or indication of endocrine disruption change at adjusted dietary concentrations of 500, 1000 or 2500 ppm. 

Organ Weights

Intergroup differences in organ weights did not indicate any effect of test item exposure at adjusted dietary concentrations of 500, 1000 or 2500 ppm.

Histopathology

No findings which could be related to the administration of the test item were apparent in the tissues examined

Conclusion

Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for the adult animals was considered to be the adjusted dietary concentration of 2500 ppm (the highest dosage tested). The No Observed Adverse Effect Level for reproduction and growth, development and survival of the offspring was considered to be the adjusted dietary concentration of 2500 ppm.