Disodium oxybis[methylbenzenesulphonate]

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 24 November 2000

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147

Version / remarks:

24 November 2000

Principles of method if other than guideline:

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 250, 500, and 1000 mg/kg bw/day when adjusted for purity of 83.9%.
A further group of twenty-four time mated females was exposed to the vehicle only (Distilled water) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study.

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Disodium oxybis[methylbenzenesulphonate]

EC Number:

277-242-0

EC Name:

Disodium oxybis[methylbenzenesulphonate]

Cas Number:

73037-34-0

Molecular formula:

C14H12Na2O7S2

IUPAC Name:

disodium oxybis(methylbenzenesulfonate)

Test material form:

solid

Details on test material:

Batch No. RJE 74440

Specific details on test material used for the study:

Identification : Ditolylether disulfonic acid disodium salt, isomer mixture (CAS No 73037-34-0)
Physical State/Appearance : Solid, slight beige powder
Composition of test item : The test item is an “UVCB substance”, consisting of Ditolylether trisulfonic acid trisodium salt, isomer mixture 27.7%, Ditolylether disulfonic acid disodium salt, isomer mixture 55.6% and Ditolylether sulfonic acid sodium salt, isomer mixture 0.6%.
Purity : 83.9%
Batch Number : RJE 74440
Storage Conditions : Ambient temperature, in the dark

Test animals

Species:

rat

Strain:

other: Sprague- Dawley Crl:CD® (SD) IGS BR

Details on test animals or test system and environmental conditions:

Animal Information
A total of ninety-six time-mated female Sprague-Dawley CCrl:CD® (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were received in two deliveries two days apart. The first delivery consisted of two batches of animals at Day1 and Day 0 respectively, the second delivery consisted of one batch of animals at Day 1 of gestation. The day that positive evidence of mating (sperm in the vaginal smear) was observed at the animal supplier was designated Day 0 of gestation. At the start of treatment (Day 5 of gestation) the females weighed 202 to 307g.

Animal Care and Husbandry
The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). 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. A certificate of analysis of the batch of diet used is given in Annex 2. 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). The diet, drinking water, bedding and environmental enrichment was 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 mean temperatures and humidity are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20% respectively; there were no deviations from these targets.
The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Four dose groups (control, low, intermediate and high) each comprising 24 time-mated females (expected to achieve 20 pregnant females per group) were used. For consistency with other toxicity work, including a preliminary pre-natal study, the test item was formulated in distilled water. Dose levels were based on available toxicity data including a preliminary pre-natal study, where dosages up to 1000 mg/kg bw/day had been well tolerated. Dosage levels of 0 (Control), 250, 500 and 1000 mg/kg bw/day at a dose volume of 5 ml/kg were therefore selected, in collaboration with the sponsor, based on the available toxicological data.
Dosages were adjusted for a purity of 83.9%.

The test item was administered daily, from Day 5 to Day 19 of gestation inclusive, by gavage. Control animals were treated in an identical manner with the vehicle alone.

The study was performed between 25 November 2015 and 20 April 2016.
The in-life phase of the study was conducted between 29 November 2015 (first day of treatment) and 15 December 2015 (final day of necropsy).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in Distilled Water. On each day of formulation preparation, for each concentration the required amount of Test Item was weighed out and added to a volume of vehicle.
Additional vehicle was added to the resulting formulation to make it up to the required volume and shaken/mixed to give a homogeneous bulk formulation. These bulk formulations were subsequently divided into the required daily aliquots and stored at 4°C in the dark until the day of use.

The stability and homogeneity of the test item formulations were previously determined by Envigo Research Limited, Shardlow, UK Analytical Services and showed that Test Item formulations were stable for at least twenty-five days when stored at 4°C in the dark. Formulations for this main study were therefore made in two batches ensuring that all formulations were used within the known stability period.
On both formulation occasions, representative samples of dosing formulations were analyzed for concentration of Ditolylether disulfonic acid disodium salt, isomer mixture (CAS No 73037-34-0) at Envigo Research Limited Analytical Laboratory, Shardlow.

The results indicate that the prepared formulations were within 100% to 106% of nominal concentration confirming the accuracy of the preparation procedure.

Details on mating procedure:

A total of ninety-six time-mated female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were received in two deliveries two days apart. The first delivery consisted of two batches of animals at Day1 and Day 0 respectively, the second delivery consisted of one batch of animals at Day 1 of gestation. The day that positive evidence of mating (sperm in the vaginal smear) was observed at the animal supplier was designated Day 0 of gestation. At the start of treatment (Day 5 of gestation) the females weighed 202 to 307g.

Duration of treatment / exposure:

The test item was administered daily, from Day 5 to Day 19 of gestation inclusive, by gavage. Control animals were treated in an identical manner with the vehicle alone.

Frequency of treatment:

daily

Duration of test:

All animals were killed on Day 20 of gestation.

Doses / concentrations

Remarks:

0(control), 250, 500 or 1000 mg/kg bw/day adjusted for purity of 83.9%

No. of animals per sex per dose:

24 female animals per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Clinical Observations
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for
animals at terminal kill (Day 20).

Food Consumption
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

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

Terminal Investigation
Necropsy
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight
The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.
Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths (one fetus from litter 42 (250 mg/kg bw/day) and one fetus from litter 74 (1000 mg/kg bw/day)) for reporting purposes
All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):

Left Horn Cervix Right Horn

L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16

V = viable fetus

The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for either skeletal alterations (skeletal examinations) or soft tissue alterations (visceral examinations).

Visceral Examinations
At necropsy, alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. These fetuses were subsequently transferred to distilled water prior to examination
for visceral anomalies under a low power binocular microscope and following examination transferred to 10% Buffered Formalin for storage.
Visceral examinations commenced with an external assessment of general appearance, including the limbs, digits, genitals, anus, tail and umbilicus. Once completed the head, and
subsequently the tongue was removed, for examination of the tongue, palate and surrounding tissue including the teeth, genioglossus and nasopharynx. Serial sections were made of the head and lower jaw to enable a detail examination of brain morphology and the lower jaw was also sectioned to allow examination of the incisors, multicuspid teeth and genioglossus muscle.
The skin was opened up to allow further visceral examination and the internal sex of the fetuses was confirmed. The position of the umbilical artery and the liver, stomach spleen, pancreas and intestines assessed. These tissues were then removed to enable examination of the under lying abdominal tissues including the kidneys which were transversely cut to enable assessment of the cortex, medulla and papilla.
Following these examinations the thoracic tissues were examined, commencing first with the diaphragm lungs, azygo us vein and thymus. The lungs and thymus were subsequently removed to allow assessment of the heart and cervicothoracic blood vessels. Examination of the heart included the external size and shape of the ventricles and atria, the entry and exit of the blood vessels around the heart and assessment of the foramen ovale, atrio-ventricular valves, semi-lunar valves, ventricular septum and general proportions of ventricular walls and cavities.

Skeletal Examinations
At necropsy, fetuses not allocated to visceral examinations were identified using cardboard tags marked with chinagraph pencil and placed in 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and alterations and storage.
For assessment, fetuses were placed in a petri dish containing glycerol and examined using a microscope. Fetuses were examined whole but for evaluation and reporting purposes the skeleton was divided into the following regions: skull, vertebral column, ribs, sternebrae, pectoral girdle, pelvic girdle, fore limbs and hind limbs. A peer review (approximately 10% of the total number of litters examined) was performed as part of the overall skeletal examination procedures for the study.

Data Evaluation
All data was summarized in tabular form, including reproductive indices. Group mean values were calculated to include data from all females with live fetuses on Day 20 of gestation.
Values given in appendices may represent rounded values for presentation purposes. Group mean values were generally calculated using unrounded values therefore it is not always possible to calculate the exact group mean values from values presented in the appendices.
As the litter was standard unit of assessment, values were first calculated within the litter and group mean values represent the mean of these individual litter values.
Food conversion efficiency was calculated for each group during the first two weeks of gestation. It was not calculated during the final week of gestation as body weight values were increasingly affected by the contribution of the litter, which is influenced by litter size as well as fetal growth.

Pre and Post Implantation Loss
Percentage pre-implantation loss was calculated as:
[(number of corpora lutea - number of implantations) : (number of corpora lutea)] x 100

Percentage post-implantation loss was calculated as:
[(number of implantations - number of live fetuses) : (number of implantations)] x 100

Sex Ratio
Sex ratio was calculated as:
% male fetuses (sex ratio) = [(Total number of fetuses : Number of male fetuses)] x 100

Statistical Analysis
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Body weight and body weight change (including adjustment for the contribution of the gravid uterus), food consumption, gravid uterus weight, litter data and fetal, litter and placental weights and external, visceral and skeletal observations.
Data were first analysed using Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance. Where there was no significance, parametric methodology was
applied using one way analysis of variance and, if significant, Dunnett’s multiple comparison test. Where statistical significance was observed, non parametric methodology was applied using Kruskal-Wallis nonparametric analysis of variance; and, if significant, pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test. Due to the non-normal distribution of the data, external, visceral and skeletal observations were generally analyzed using non-parametric methodology.

ARCHIVING
Records and documentation relating to this study (including electronic records) will be maintained in the archives of Envigo Research Limited, Shardlow, Derbyshire for a period of ten years from the date on which the Study Director signs the final report.

Examinations

Maternal examinations:

Clinical Observations
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing
period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for
animals at terminal kill (Day 20).

Food Consumption
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

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

Ovaries and uterine content:

Necropsy
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal
examination and any macroscopic abnormalities were recorded. The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight
The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.
Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths (one fetus from litter 42 (250 mg/kg bw/day) and one fetus from litter 74
(1000 mg/kg bw/day)) for reporting purposes
All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):

Left Horn Cervix Right Horn

L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16

V = viable fetus

The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for either skeletal
alterations (skeletal examinations) or soft tissue alterations (visceral examinations).

Fetal examinations:

Visceral Examinations
At necropsy, alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. These fetuses were subsequently transferred to distilled water prior to examination
for visceral anomalies under a low power binocular microscope and following examination transferred to 10% Buffered Formalin for storage.
Visceral examinations commenced with an external assessment of general appearance, including the limbs, digits, genitals, anus, tail and umbilicus. Once completed the head, and
subsequently the tongue was removed, for examination of the tongue, palate and surrounding tissue including the teeth, genioglossus and nasopharynx. Serial sections were made of the
head and lower jaw to enable a detail examination of brain morphology and the lower jaw was also sectioned to allow examination of the incisors, multicuspid teeth and genioglossus
muscle.
The skin was opened up to allow further visceral examination and the internal sex of the fetuses was confirmed. The position of the umbilical artery and the liver, stomach spleen,
pancreas and intestines assessed. These tissues were then removed to enable examination of the under lying abdominal tissues including the kidneys which were transversely cut to
enable assessment of the cortex, medulla and papilla.
Following these examinations the thoracic tissues were examined, commencing first with the diaphragm lungs, azygo us vein and thymus. The lungs and thymus were subsequently
removed to allow assessment of the heart and cervicothoracic blood vessels. Examination of the heart included the external size and shape of the ventricles and atria, the entry and exit of
the blood vessels around the heart and assessment of the foramen ovale, atrio-ventricular valves, semi-lunar valves, ventricular septum and general proportions of ventricular walls and
cavities.

Skeletal Examinations
At necropsy, fetuses not allocated to visceral examinations were identified using cardboard tags marked with chinagraph pencil and placed in 70% IMS in distilled water. The fetuses
were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and alterations
and storage.
For assessment, fetuses were placed in a petri dish containing glycerol and examined using a microscope. Fetuses were examined whole but for evaluation and reporting purposes the
skeleton was divided into the following regions: skull, vertebral column, ribs, sternebrae, pectoral girdle, pelvic girdle, fore limbs and hind limbs. A peer review (approximately 10%
of the total number of litters examined) was performed as part of the overall skeletal examination procedures for the study.

Statistics:

The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Body weight and body weight change (including adjustment for the contribution of the gravid uterus), food consumption, gravid uterus weight, litter data and fetal, litter and placental
weights and external, visceral and skeletal observations.
Data were first analysed using Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance. Where there was no significance, parametric methodology was
applied using one way analysis of variance and, if significant, Dunnett’s multiple comparison
test. Where statistical significance was observed, non parametric methodology was applied using Kruskal-Wallis nonparametric analysis of variance; and, if significant, pairwise
analysis of control values against treated values using the Mann-Whitney ‘U’ test. Due to the non-normal distribution of the data, external, visceral and skeletal observations were
generally analyzed using non-parametric methodology.

Indices:

Pre and Post Implantation Loss
Percentage pre-implantation loss was calculated as:
[(number of corpora lutea - number of implantations) : (number of corpora lutea)] x 100

Percentage post-implantation loss was calculated as:
[(number of implantations - number of live fetuses) : (number of implantations)] x 100

Sex Ratio
Sex ratio was calculated as:
% male fetuses (sex ratio) = [(Total number of fetuses : Number of male fetuses)] x 100

Historical control data:

Historical control data are available for:
Normal Range Data for Pre-Natal Study Gestation Body Weights in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Normal Range Data for Pre-Natal Study Gestation Food Consumption in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Normal Range Data for Pre-Natal Litter Data in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Normal Range Data for Pre-Natal Study External Fetal Observations in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Normal Ranges for Pre-Natal Study Visceral Fetal Findings in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Normal Ranges for Pre-Natal Study Skeletal Fetal Findings in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs did not indicate any effect of treatment at 250, 500 or 1000 mg/kg bw/day.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and body weight gain during gestation were unaffected by treatment at 250, 500 or 1000 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption during gestation was unaffected by treatment at 250, 500 or 1000 mg/kg bw/day.

Food efficiency:

no effects observed

Description (incidence and severity):

Food conversion efficiency was unaffected by treatment 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 did not reveal any effect of treatment at 250, 500 or 1000 mg/kg bw/day.

Ophthalmological findings:

not examined

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:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic necropsy findings did not indicate any effect of treatment at 250, 500 or 1000 mg/kg bw/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The number of corpora lutea and pre-implantation losses (events established prior to treatment) were generally similar in all groups. At 500 mg/kg bw/day, pre-implantation loss was marginally higher than control but this was principally due to two litters that showed atypically high pre-implantation loss. As neither litter showed any subsequent postimplantation loss, it was considered that these litters had no adverse influence on the assessment of test material effects on litter and fetal parameters.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No total litter loss by resorption was observed.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions (including dead fetuses), live litter size and sex ratio at 250, 500 or 1000 mg/kg bw/day.

Dead fetuses:

no effects observed

Description (incidence and severity):

A fetus that had died shortly before necropsy. These were included as late deaths (one fetus from litter 42 (250 mg/kg bw/day) and one fetus from litter 74 (1000 mg/kg bw/day)) for reporting purposes.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

All pregnant females successfully maintained their litter to Day 20 of gestation; there were no incidences of total litter loss by resorption during the study.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): All pregnant females successfully maintained their litter to Day 20 of gestation; there were no incidences of total litter loss by resorption during the study.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All pregnant females successfully maintained their litter to Day 20 of gestation; there were no incidences of total litter loss by resorption during the study based on the 23, 23, 23 and 24 females with live young at Day 20 of gestation at 0 (control), 250, 500 and 1000 mg/kg bw/day respectively.

Other effects:

no effects observed

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

food efficiency

gross pathology

maternal abnormalities

mortality

necropsy findings

number of abortions

pre and post implantation loss

total litter losses by resorption

water consumption and compound intake

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean fetal, litter weights were unaffected by maternal treatment at 250, 500 or 1000 mg/kg bw/day.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Numbers of implantations, in-utero offspring survival, live litter size and sex ratio were unaffected by maternal treatment at 250, 500 or 1000 mg/kg bw/day.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Numbers of implantations, in-utero offspring survival, live litter size and sex ratio were unaffected by maternal treatment at 250, 500 or 1000 mg/kg bw/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Mean fetal, litter weights and litter size were unaffected by maternal treatment at 250, 500 or 1000 mg/kg bw/day.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

External fetal findings did not indicate any effect of maternal treatment at 250, 500 or 1000 mg/kg bw/day.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Skeletal fetal findings did not indicate any effect of maternal treatment at 250, 500 or 1000 mg/kg bw/day.

Visceral malformations:

no effects observed

Description (incidence and severity):

Visceral fetal findings did not indicate any effect of maternal treatment at 250, 500 or 1000 mg/kg bw/day.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Based on the results of the study, the No Observed Effect Level (NOEL) for the pregnant female rat and the survival, growth and development of the conceptus was 1000 mg/kg bw/day (limit dose, highest dose tested).

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

other: 1000 mg/kg bw/day (limit dose, highest dose tested).

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Relevant for humans:

yes

Any other information on results incl. tables

Methods

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 250, 500, and 1000 mg/kg bw/day when adjusted for purity of 83.9%. A further group of twenty-four time mated females was exposed to the vehicle only (Distilled water) to serve as a control.

 

Clinical signs, body weight change, food and water consumptions were monitored during the study.

 

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

 

Results

Mortality

There were no unscheduled deaths during the study.

 

Clinical Observations

Clinical signs did not indicate any effect of treatment at 250, 500 or 1000 mg/kg bw/day.

 

Body Weight

Body weight and body weight gain during gestation were unaffected by treatment at 250, 500 or 1000 mg/kg bw/day.

 

Food Consumption

Food consumption during gestation was unaffected by treatment at 250, 500 or 1000 mg/kg bw/day.

 

Food Conversion Efficiency

Food conversion efficiency was unaffected by treatment at 250, 500 or 1000 mg/kg bw/day.

 

Water Consumption

Visual inspection of water bottles did not reveal any effect of treatment at 250, 500 or 1000 mg/kg bw/day.

 

Post Mortem Studies

Macroscopic necropsy findings did not indicate any effect of treatment at 250, 500 or 1000 mg/kg bw/day.

Litter Data

Numbers of implantations, in-utero offspring survival, live litter size and sex ratio were unaffected by maternal treatment at 250, 500 or 1000 mg/kg bw/day. Litter Placental and Fetal Weights Mean fetal, litter or placental weights were unaffected by maternal treatment at 250, 500 or 1000 mg/kg bw/day.

 

Fetal Examination

External Examinations

External fetal findings did not indicate any effect of maternal treatment at 250, 500 or 1000 mg/kg bw/day.

 

Detailed Visceral Examinations

Visceral fetal findings did not indicate any effect of maternal treatment at 250, 500 or 1000 mg/kg bw/day.

 

Detailed Skeletal Examinations

Skeletal fetal findings did not indicate any effect of maternal treatment at 250, 500 or 1000 mg/kg bw/day.

Applicant's summary and conclusion

Conclusions:

Based on the results of the study, the No Observed Effect Level (NOEL) for the pregnant female rat and the survival, growth and development of the conceptus was 1000 mg/kg bw/day (limit dose, highest dose tested).

Executive summary:

Methods

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 250, 500, and 1000 mg/kg bw/day when adjusted for purity of 83.9%. A further group of twenty-four time mated females was exposed to the vehicle only (Distilled water) to serve as a control.

 

Clinical signs, body weight change, food and water consumptions were monitored during the study.

 

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

 

Results

Mortality

There were no unscheduled deaths during the study.

 

Clinical Observations

Clinical signs did not indicate any effect of treatment at 250, 500 or 1000 mg/kg bw/day.

 

Body Weight

Body weight and body weight gain during gestation were unaffected by treatment at 250, 500 or 1000 mg/kg bw/day.

 

Food Consumption

Food consumption during gestation was unaffected by treatment at 250, 500 or 1000 mg/kg bw/day.

 

Food Conversion Efficiency

Food conversion efficiency was unaffected by treatment at 250, 500 or 1000 mg/kg bw/day.

 

Water Consumption

Visual inspection of water bottles did not reveal any effect of treatment at 250, 500 or 1000 mg/kg bw/day.

 

Post Mortem Studies

Macroscopic necropsy findings did not indicate any effect of treatment at 250, 500 or 1000 mg/kg bw/day.

Litter Data

Numbers of implantations, in-utero offspring survival, live litter size and sex ratio were unaffected by maternal treatment at 250, 500 or 1000 mg/kg bw/day. Litter Placental and Fetal Weights Mean fetal, litter or placental weights were unaffected by maternal treatment at 250, 500 or 1000 mg/kg bw/day.

 

Fetal Examination

External Examinations

External fetal findings did not indicate any effect of maternal treatment at 250, 500 or 1000 mg/kg bw/day.

 

Detailed Visceral Examinations

Visceral fetal findings did not indicate any effect of maternal treatment at 250, 500 or 1000 mg/kg bw/day.

 

Detailed Skeletal Examinations

Skeletal fetal findings did not indicate any effect of maternal treatment at 250, 500 or 1000 mg/kg bw/day.

 

Conclusion

Based on the results of the study, the No Observed Effect Level (NOEL) for the pregnant female rat and the survival, growth and development of the conceptus was 1000 mg/kg bw/day (limit dose, highest dose tested).