C16-18 (evennumbered, C18 unsaturated) alkyl bis(2-hydroxyethyl) amine oxide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 27 September 2017 Experimental Completion Date: date of final thyroid hormone report

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:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 29 July 2016).

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethanol, 2,2'-iminobis-, N-tallow alkyl derivs., N-oxides

EC Number:

263-179-6

EC Name:

Ethanol, 2,2'-iminobis-, N-tallow alkyl derivs., N-oxides

Cas Number:

61791-46-6

Details on test material:

The test material was identified by name and specifications:
- chemical name: Tallowbis(2-hydroxyethyl)amine oxide
- cas nr : 61791-46-6
- batch no: 90.2 7 (CoA attached to the report):
Amine oxide : 50.1 %
water: 25% (not on CoA)
Diethyleneglycol: 25% (not on CoA)
Free amine : 0.55%
Free peroxide: 0.17% on CoA,but 0.07% in report text
pH (10+90 water): 7.1

Color : passes test

It was supplied by the study sponsor on 30 July 1990. Stored in the dark at room temperature.

Specific details on test material used for the study:

Identification : Tallow bis(2-hydroxyethyl)amineoxide
Physical State/Appearance : Extremely pale yellow paste
CAS Number : 61791-46-6
Purity : 50.66% (UVCB)
Batch Number : 1548134
Date Received : 17 May 2017
Storage Conditions : Ambient temperature and humidity in darkness; used/formulated in light
Expiry Date : 27 March 2018
Dosages were adjusted for the purity (UVCB content) of the Test Item of 50.66%.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for nineteen days during which time their health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 277 to 358g, and were approximately eleven weeks old. The females weighed 197 to 250g, 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 pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Annex 6. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during the final week of gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Distilled water. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results showed the formulations to be stable for at least eleven days when stored refrigerated. Formulations were therefore prepared weekly and stored at approximately 4°C in the dark.

Samples of the test item formulations were taken on three occasions and analyzed for concentration of Tallow bis(2-hydroxyethyl)amineoxide at Envigo Research Limited, Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Annex 2. The results indicate that the prepared formulations were within 89-104% of the nominal concentration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Preparation of calibration standards
Stock solutions of test item in dilution solvent were prepared for external standard calibration. An aliquot, approximately 0.1 g of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with dilution solvent to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration of 0.2 mf/mL.

On each occasion, standard solutions derived from two stock standard solutions wre used for calculation.

Calibration solutions were injected into the instrument, at the beginning and end of each sample analysis as a minimum, using the conditins detailed in the instrument parameters described below.

To assess the calibration range of the method, a range of standard solutions were prepared in dilution solvent from a stock solutiopn of 1 mg/mL by serial dilution covering the concentration range 0.1029 mg/mL to 0.4116 mg/mL.

Preparation of test samples
The formulations received were extracted with dilution solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with dilution solvent , then ultra-sonicated for 15 minutes. Where necessary, sample solutions were further diluted with dilution solvent to achieve the working concentration.

Preparation of accuract and precision samples
Samples of diltilled water were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis.

The concentration of Test Item in the final solution was quantified by gas chromatography using flame ionisation detection.

Instrument parameters
GC system: Agilent Technologies 6890, incorporating autosampler and workstation
Column: Rxi-5ms (15 m x 0.25 mm idf x 0.25 µm film)
Oven temperature programme: Oven - 100°C for 0 minutes with 10°C/minute to 300°C for 0 minutes
Injection temperature: 250°C
Flame ionisation detection temperature: 300°C
Injection volume: 1.0 µL
Retention time: ~2-13 mins

Details on mating procedure:

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.

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

Duration of test:

approximately six weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Remarks:

Concentration adjusted for purity ~ 2.5 mg/mL

Doses / concentrations 2

Dose / conc.:

75 mg/kg bw/day (actual dose received)

Remarks:

Concentration adjusted for purity: 7.5 mg/mL

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Remarks:

Following body weight losses for both sexes at 200 mg/kg bw/day, the dosage was reduced to 150 mg/kg bw/day from Day 10 of dosing.

No. of animals per sex per dose:

12 males/ 12 females per dose group and control group

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 GV48NN). In this range-finding study a dosage of 250 mg/kg bw/day was concluded to be too high for more long term investigation but no effects were apparent at a dosage of 150 mg/kg bw/day. A dosage of 200 mg/kg bw/day was therefore selected as the high dosage for this OECD 421 study, with low and intermediates dosage of 25 and 75 mg/kg bw/day respectively. Following body weight losses for both sexes at 200 mg/kg bw/day, the dosage was reduced to 150 mg/kg bw/day from the 25 October 2017 (Day 10 of dosing). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

Examinations

Maternal 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 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.

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

Estrous Cycle Assessment
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.

Terminal Investigations
Necropsy
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, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. Examination of offspring was restricted to a macroscopic external examination except where abnormalities were observed; then an additional internal examination was performed.

Thyroid Hormone Analysis
Blood samples taken to produce serum were allowed to clot, centrifuged and the serum from each blood sample stored frozen at lower than -60ºC. Blood samples taken to produce plasma were collected into K2EDTA, centrifuged, and the plasma from each blood sample stored frozen at lower than -60ºC. Samples were taken as follows:

Where possible serum samples were taken from two randomly allocated offspring from each litter on Day 4 post partum (females where possible to maximize the number of males available for Day 13 post partum visible nipple counts; 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.
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. 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 after being placed in fixative (partial fixation).

Histopathology
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:
Epididymides♦
Glans penis
Seminal Vesicles (with coagulating gland)
Gross lesions
LABC (levator ani-bulbocavernous) muscle
Thyroid/Parathyroid
Mammary gland
Uterus/Cervix (with oviducts)
Ovaries
Vagina
Pituitary

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

All tissues were dispatched to the histology processing Test Site (Propath UK Ltd., Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU, United Kingdom) for processing. The tissues from control and 200/150 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.

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.

Pathology
Microscopic examination was conducted by the Study Pathologist. A peer review of the findings observed was conducted at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS.

Ovaries and uterine content:

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

Fetal examinations:

Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum
iii. Sex of offspring on Days 1, 4 and 13 post partum
iv. Clinical condition of offspring from birth to Day 13 post partum
v. Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data)

Physical Development
All live offspring were assessed for ano-genital distance on Day 1 post partum. Additionally, visible nipple count was performed for all male offspring on Day 13 post partum.

Statistics:

Please refer to "Any other information on materials and methods including tables"

Indices:

Please refer to "Any other information on materials and methods including tables"

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed that indicated any systemic effect of treatment at dosage of 25, 75 or 200/150 mg/kg bw/day.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was one unscheduled death on the study. Control female 14 was found dead during late gestation on Day 36 of the study. Although no clinical signs had been apparent prior to death, dead fetuses were found in both uterine horns at necropsy and the vagina was filled with red fluid. Additionally the liver, adrenals and spleen were noted to be enlarged at necropsy. Histopathology findings showed the animal was found to have a malignant lymphoma which was considered to be the cause of death.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg bw/day mean body weight loss was apparent for both sexes during the first week of treatment. This dosage was reduced to 150 mg/kg bw/day during Week 2 (Day 10). Recovery of body weight gain was apparent for females, with gain being statistically significantly higher than controls during Week 2, and remaining similar to controls throughout gestation and lactation. Males also showed recovery of body weight gain, although gain remained statistically significantly lower than controls during Week 2. While body weight gain for males during Week 3 was superior to control, subsequent body weight gain to termination was lower than control (attaining statistical significance during Weeks 4 and 5). Overall body weight gain for males was statistically significantly lower than control at termination. Mean bodyweights for males were lower than controls from Week 2 onwards, with differences frequently attaining statistical significance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 200/150 mg/kg bw/day, food consumption for females was lower than control during the first week of the study. Food consumption remained lower than control during the second week of treatment but, at the level observed this could not be clearly attributed to treatment.
There was no effect of treatment on food consumption of females during the gestation or lactation phases of the study at 25, 75 or 200/150 mg/kg bw/day.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

The value of food conversion efficiency where animal have shown a group mean body weight loss is equivocal. At 200/150 mg/kg bw/day, for both sexes during the first week of treatment and males during the second week of treatment, food conversion efficiency was inferior to concurrent control, reflecting the initial mean body weight loss for both sexes and subsequent lower bodyweight gains for males.
Food conversion efficiency for both sexes treated with 25 or 75 mg/kg bw/day showed no effect of treatment.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

There was no effect of treatment on water consumption for either sex at 25, 75 or 200/150 mg/kg bw/day; daily visual inspection of water bottles revealed no overt intergroup differences.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 25, 75 or 200/150 mg/kg bw/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no statistically significant differences from control for thyroid/parathyroid weights of either sex at 25, 75 or 200/150 mg/kg bw/day.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence, type and distribution of macroscopic findings observed at terminal necropsy of surviving animals did not indicate any effect of treatment at dosages of 25, 75 or 200/150 mg/kg bw/day.

One female treated at 25 mg/kg bw/day was observed to have an enlarged spleen, but in the absence of any similar findings at 75 or 200/150 mg/kg bw/day, this is considered to be incidental and unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathological examination of reproductive tissues from the control and 200/150 mg/kg bw/day animals did not reveal any findings considered to be related to treatment with the test item. In particular there were no consistent treatment-related pathologic findings in the testes following the qualitative examination of the stages of spermatogenesis in the testes (no treatment-related related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or in the ovaries following the evaluation of the follicles and corpora lutea.

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

Not applicable, as rats do not abort.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Group 1: Mean post-implantation loss - 3.7%
Group 2: 5.7%
Group 3: 4.6%
Group 4: 6.4%

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

1 animal in the control group and 1 in the low dose group: No pups observed to be born but implantation in uterus

Early or late resorptions:

no effects observed

Description (incidence and severity):

1 animal in the control group and 1 in the low dose group: No pups observed to be born but implantation in uterus

Dead fetuses:

no effects observed

Description (incidence and severity):

Live birth Index
Group 1: 98.3%
Group 2: 99.2%
Group 3: 100%
Group 4: 99.2%

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Surviving to terminal necropsy
100% across all dose groups

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Results (fetuses)

Fetal body 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 25, 75 or 200/150 mg/kg bw/day.
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 25, 75 or 200/150 mg/kg bw/day.
Clinical signs apparent for the offspring during the study were generally typical of the age observed and neither the distribution nor incidence of these findings indicated any effect of maternal treatment.

Reduction in number of live offspring:

no effects observed

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 25, 75 or 200/150 mg/kg bw/day.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio for the offspring was similar to control in all treated groups and did not indicate any selective effect of maternal treatment on survival for either sex at any of the dosages investigated.

Changes in litter size and weights:

no effects observed

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 25, 75 or 200/150 mg/kg bw/day.

Changes in postnatal survival:

no effects observed

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 25, 75 or 200/150 mg/kg bw/day.

External malformations:

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 systemic effect of maternal treatment at 25, 75 or 200/150 mg/kg bw/day.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Effect levels (fetuses)

Key result

Dose descriptor:

NOEL

Effect level:

>= 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: were no effects apparent for reproduction or for the growth, development and survival of the offspring at any of the dosages tested

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

The oral administration of Tallow bis(2-hydroxyethyl)amineoxide to rats by gavage, at a dose level of 200 mg/kg bw/day, resulted in body weight losses for both sexes. Despite the lowering of the high dosage to 150 mg/kg bw/day, lower body weight gain in males was apparent, the extent of which was considered to preclude this dosage from being classified as a No Observed Adverse Effect Level (NOAEL).

Other than increased post-dosing salivation, there were no treatment-related findings apparent for either sex at 75 mg/kg bw/day and therefore this dosage is considered to be the NOAEL for the adult animals. There were no effects apparent for reproduction or for the growth, development and survival of the offspring at any of the dosages tested therefore the No Observed Effect Level (NOEL) for reproduction and for the growth, development and survival of the offspring was considered to be at least 150 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 six weeks (males) and eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 25, 75 and 200 mg/kg bw/day (reduced to 150 mg/kg bw/day on Day 10) (incorporating a correction factor for 50.66% purity). A control group of twelve males and twelve females was dosed with vehicle alone (Distilled water) 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 external observations were detected an internal necropsy was performed.

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 was one unscheduled death on the study. Control female 14 was found dead during late gestation on Day 36 of the study. Although no clinical signs had been apparent prior to death, dead fetuses were found in both uterine horns at necropsy and the vagina was filled with red fluid. Additionally the liver, adrenals and spleen were noted to be enlarged at necropsy. Histopathology findings showed the animal was found to have a malignant lymphoma which was considered to be the cause of death.

Clinical Observations

There were no clinical signs observed that indicated any systemic effect of treatment at dosage of 25, 75 or 200/150 mg/kg bw/day.

Body Weight

At 200 mg/kg bw/day mean body weight loss was apparent for both sexes during the first week of treatment. This dosage was reduced to 150 mg/kg bw/day during Week 2 (Day 10). Subsequent recovery of body weight gain was apparent for females, becoming similar to controls throughout gestation and lactation. Males also showed recovery of body weight gain, although gain remained lower than controls during Week 2. While body weight gain for males during Week 3 was superior to control, subsequent body weight gain to termination was lower than control and overall body weight gain for males was statistically significantly lower than control at termination. Mean body weights for males were lower than controls from Week 2 onwards, with differences frequently attaining statistical significance.

There was no effect of treatment on body weight or body weight gains for either sex at 25 or 75 mg/kg bw/day throughout the study.

Food Consumption& Food Conversion Efficiency

At 200/150 mg/kg bw/day, food consumption for males was lower than control throughout the pre-pairing and post-pairing phases of the study.

At 200/150 mg/kg bw/day, food consumption for females was lower than control during both weeks of the study. 

There was no effect of treatment on food consumption of females during the gestation or lactation phases of the study at 25, 75 or 200/150 mg/kg bw/day.

At 200/150 mg/kg bw/day, for both sexes during the first week of treatment and males during the second week of treatment, food conversion efficiency was inferior to concurrent control.

Food conversion efficiency for both sexes treated with 25 or 75 mg/kg bw/day showed no effect of treatment.

Water Consumption

There was no effect of treatment on water consumption for either sex at 25, 75 or 200/150 mg/kg bw/day.

Reproductive Performance

Estrous Cycle

There was no effect of treatment on estrous cycling at 25, 75 or 200/150 mg/kg bw/day.

Mating

There was no effect of treatment on mating performance at 25, 75 or 200/150 mg/kg bw/day.

Fertility

There was no effect of treatment on fertility at 25, 75 or 200/150 mg/kg bw/day.

Gestation Length

There was no effect of treatment on gestation length at 25, 75 or 200/150 mg/kg bw/day.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no effect of maternal treatment on the numbers of implantations, post-implantation loss, litter size at birth/Day 1, subsequent offspring survival to Day 13 of age or sex ratios at 25, 75 or 200/150 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 25, 75 or 200/150 mg/kg bw/day. There was no effect of treatment on ano-genital distance for offspring on Day 1post partumand visible nipple count for male offspring on Day 13post partumat 25, 75 or 200/150 mg/kg bw/day.

Offspring Observations

Clinical signs apparent for the offspring during the study were generally typical of the age observed and neither the distribution nor incidence of these findings indicated any effect of maternal treatment.

Pathology

Necropsy

Offspring

Macroscopic necropsy findings for offspring on the study did not indicate any systemic effect of maternal treatment at 25, 75 or 200/150 mg/kg bw/day.

Adults

Macroscopic necropsy findings for adult animals on the study did not indicate any effect of treatment at 25, 75 or 200/150 mg/kg bw/day.

Thyroid Hormone Analysis

Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 25, 75 or 200/150 mg/kg bw/day.

Organ Weights

There were no statistically significant differences from control for thyroid/parathyroid weights of either sex at 25, 75 or 200/150 mg/kg bw/day.

There was no adverse effect of treatment on male reproductive organ weights at 25, 75 or 200/150 mg/kg bw/day.

Histopathology

Histopathological examination of reproductive tissues from 200/150 mg/kg bw/day animals did not reveal any findings considered to be related to treatment with the test item.

Conclusion

The oral administration of Tallow bis(2-hydroxyethyl)amineoxide to rats by gavage, at a dose level of 200 mg/kg bw/day, resulted in body weight losses for both sexes. Despite the lowering of the high dosage to 150 mg/kg bw/day, lower body weight gain in males was apparent, the extent of which was considered to preclude this dosage from being classified as a No Observed Adverse Effect Level (NOAEL). 

Other than increased post-dosing salivation, there were no treatment-related findings apparent for either sex at 75 mg/kg bw/day and therefore this dosage is considered to be the NOAEL for the adult animals. There were no effects apparent for reproduction or for the growth, development and survival of the offspring at any of the dosages tested therefore the No Observed Effect Level (NOEL) for reproduction and for the growth, development and survival of the offspring was considered to be at least 150 mg/kg bw/day.