Oligomerisation products of ethylene oxide with reaction products of rape oil and ethanolamine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 09 April 2009 and 30 July 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of GLP inspection: 19/08/2008 Date of Signature on GLP certificate: 04/03/2009

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

Description : yellow liquid
Date received : 08 April 2009
Storage conditions : room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: A sufficient number of male and female Wistar Han™:HsdRccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd.
- Age at study initiation: (P) approximately 12 weeks.
- Weight at study initiation: (P) Males: 309 - 368 g; Females: 192 - 240 g.
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the mating phase, 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.
- Diet (e.g. ad libitum): The animals were allowed free access to food. A pelleted diet was used throughout the treatment period.
- Water (e.g. ad libitum): The animals were allowed free access to water. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: At least 11 days, during which their health status was assessed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature controls were set to achieve target values of 21 ± 2°C.
- Humidity (%): The relative humidity controls were set to achieve target values of 55 ± 15%.
- Air changes (per hr): At least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): Low intensity fluorescent lighting was controlled to give tweleve hours continous light and twelve hours darkness.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of the study, the test material was prepared at the appropriate concentrations as a solution in Arachis oil. The stability and homogeneity of the test material formulations were determined. Results show the formulations to be stable for at least fourteen days. Formulations were
therefore prepared weekly and stored at approximately +4ºC in the dark.



VEHICLE
- Justification for use and choice of vehicle (if other than water): The test material was prepared at the appropriate concentrations as a solution in Arachis oil. The formulations were shown to be stable for at least fourteen days.
- Amount of vehicle (if gavage): 4 ml/kg

Details on mating procedure:

- M/F ratio per cage: one male:one female basis within each dose group (during mating phase).
- Length of cohabitation: For a period of 14 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.
- Proof of pregnancy: 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 the oestrous cycle 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)
- After successful mating each pregnant female was caged (how): Females were housed individually during the period of gestation and lactation in soolid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Any other deviations from standard protocol: None.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test material formulation were taken and analysed for concentration of the substance

The concentration of the substance in the test material formulations was determined by gas chromatography (GC) using an external standard technique.

The results indicate that the prepared formulations were within 12% acceptable limits for the purpose of this study.

Please see section 'any other information on materials and methods incl. tables' for full details of GC.

Duration of treatment / exposure:

Up to fifty-five consecutive days, (including a two week maturation phase, pairing, gestation and early lactation for females)

Frequency of treatment:

The test material was administered daily.

Details on study schedule:

Chronological Sequence of Study:
i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) On Day 15, all animals were paired on a 1 male:1 female basis within each dose group for a maximum of fourteen days.
iii) 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.
iv) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.
v) The male dose groups were killed and examined macroscopically on Day 43.
vi) At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.

- Age at mating of the mated animals in the study: approximately 14 weeks.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Vehicle alone (arachis oil)
Basis:
other: a control group was dosed with vehicle alone.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
15 mg/kg/day
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
150 mg/kg/day
Basis:
nominal conc.

Doses / concentrations 4

Remarks:

Doses / Concentrations:
500 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

Groups of 10 male and 10 female animals per dose group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on previous toxicity work (28 Day Repeated Oral (Gavage) Toxicity Study in The Rat.
- Other: The test material was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 ml/kg/day of Arachis oil.
The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at regular intervals.

Positive control:

No positive control.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes (see clinical observations).


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, soon after dosing and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing and one hour after dosing at weekends (except for females during parturition where applicable).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination. Females were weighed weekly until mating was evident. Bodyweights were then recorded on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION: Yes
- During the maturation 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 surviving females with live litters, food consumption was recorded during the lactation period (Days 1 – 4).

Weekly food efficiency (bodyweight gain/food intake) was calculated retrospectively for males and for females during the pre-mating phase and during the first two weeks of gestation. Due to offspring growth and milk production, food efficiency could not be accurately calculated during the final week of gestation and during lactation.


WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes. Inter-group differences did not indicate any need for more formal gravimetric measurements.

OTHER:
Pregnancy and Parturition:
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours 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):

A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded.

Sperm parameters (parental animals):

Not examined.

Litter observations:

STANDARDISATION OF LITTERS
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded and offspring were individually identified within each litter by a tattoo on Day 1 post partum.
For each surviving litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Day 1 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring and litter weights on Days 1 and 4 post partum

Physical Development:
All live offspring were assessed for surface righting reflex on Day 1 post partum.

GROSS EXAMINATION OF DEAD PUPS:
Offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43.
- Maternal animals: Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum.

GROSS NECROPSY
- All adult animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
- For all females the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights: The epididymides and testes were removed from terminal kill adult males dissected free from fat and weighed before fixation.

Histopathology: Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where indicated:
Coagulating gland
Prostate
Epididymides *
Seminal vesicles
Gross lesions
Ovaries
Testes *
Mammary tissue (females only)
Uterus/Cervix
Pituitary
Vagina

Postmortem examinations (offspring):

SACRIFICE
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.

GROSS NECROPSY
Offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

The following parameters were subjected to statistical analysis:
Bodyweight and bodyweight change
Food consumption for females during gestation and lactation
Litter data
Sex ratio
Implantation losses and viability indices
Offspring bodyweight and bodyweight change
Offspring surface righting
Adult absolute and bodyweight relative organ weights

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 for 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:

Litter Responses:
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 5 of age).

i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
% pre-implantation loss = [(Number of Corpora Lutea - Number of implantation sites)/Number of corpora lutea] x 100

% post-implantation loss = [(Number of implantation site - Total number of offspring born)/Number of implantation sites] x 100

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

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no unscheduled deaths.
There were no toxicologically significant findings detected throughout the treatment period.
Episodes of increased salivation were evident in animals of either sex treated with 500 mg/kg/day. Isolated episodes of noisy respiration were also detected in the 500 mg/kg/day males.
Isolated episodes of increased salivation were evident in three 150 mg/kg/day males whilst a sporadic incidence of noisy respiration was observed in another male from this treatment group. A dark mass on the abdomen was noted in one 150 mg/kg/day female.
One 15 mg/kg/day male was observed with generalised scab formation.
Generalised fur loss was evident in two control females whilst generalised scab formation was seen in one control male.
Increased salivation is often recorded following the oral administration of an unpleasant tasting test material formulation and is considered not to be indicative of systemic toxicity. The remaining observations are considered incidental and not to represent an adverse effect of treatment.
No such effects were detected in females treated with 15 mg/kg/day.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males:
No adverse effect on bodyweight development was detected for treated animals, in comparison to controls.
Females:
No adverse effect on bodyweight development was detected for treated animals, in comparison to controls.


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Males:
No adverse effect on dietary intake was detected for treated animals, in comparison to controls.
Females:
No adverse effect on dietary intake was detected for treated animals, in comparison to controls.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating:
No adverse effect on mating performance was detected. All animals from the control and treated groups mated within fourteen days of pairing.

Fertility:
No adverse effect on fertility was evident in the treated animals, in comparison to controls. All treated and control females achieved pregnancy.

Gestation Length:
There was no adverse effect on gestation lengths between treatment and control females. Females from all treatment groups went on to give birth following 22 to 23.5 days of gestation.


ORGAN WEIGHTS (PARENTAL ANIMALS)
No adverse effect on organ weight measurement was detected for treated animals, in comparison to controls.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Necropsy (adults): The incidental signs detected in the adult were considered to be incidental and of no toxicological importance.

One 150 mg/kg/day female had a mass on the abdomen whilst one 15 mg/kg/day female had a mass on the left horn of the uterus. One control male had a small left testes and epididymides.
No macroscopic abnormalities were detected in the remaining animals.


HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no unscheduled deaths during the course of the study, and no female rats were found to be non-pregnant.

TREATMENT-RELATED HISTOPATHOLOGY
No treatment-related changes were observed.

OTHER HISTOPATHOLOGY
No changes occurred with sufficient incidence or variation to warrant statistical evaluation.

All morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.

PITUITARY: No significant histopathological changes were seen. Developmental cysts are a commonly observed spontaneous lesion in the rat pituitary.
TESTES/EPIDIDYMIDES: Unilateral testicular atrophy was seen in one control animal with associated reduced spermatozoal content and cellular debris in the corresponding epididymis. Spermatocoel granuloma formation was seen in one control and in one high dose animal. Such changes are commonly seen among laboratory maintained rats of this age.

SEMINAL VESICLES/COAGULATING GLANDS: No histopathological changes were observed

PROSTATE: Interstitial chronic inflammatory cell infiltrates are a commonly observed background finding in laboratory maintained rats. Prostatitis, involving infiltrates of acute inflammatory cells within the lumen of glands was also seen for one high dose animal. None of these changes were of toxicological significance.

MAMMARY GLAND: Glandular hyperplasia was observed in the mammary tissue of all female animals examined. The appearance of the mammary tissue is consistent with pregnancy and lactation.

OVARIES: No histopathological changes were seen.

UTERUS: Focal haemorrhage, fibrosis, foam cell accumulations and haemosiderin pigment deposition were all observed with varying severity in the peripheral uterine tissues of previously pregnant animals. A prominent haematoma was seen for one control animal. These changes are commonly encountered consequences of pregnancy and parturition in the female rat.

VAGINA: No pathology was observed.

Effect levels (P0)

Dose descriptor:

NOEL

Remarks:

(discriminating level)

Effect level:

>= 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Generation: Parental, no adverse effects were observed up to a dose level of 500 mg/kg bw/d

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
There were no obvious differences in the number of corpora lutea or implantation sites from treated females when compared to controls, and pre-implantation losses from treated animals were comparable to controls. Furthermore, no obvious effect on littler size, sex ration and offspring viability were detected from treatment animals, in comparison to controls.

CLINICAL SIGNS (OFFSPRING)
No adverse effect on surface righting reflex was detected in treated animals, in comparison to controls.

BODY WEIGHT (OFFSPRING)
No adverse effect on total litter weights, offspring bodyweight development was detected in treated animals, in comparison to controls.

GROSS PATHOLOGY (OFFSPRING)
The incidental signs detected in the offspring were considered to be incidental and of no toxicological importance.

The interim death offspring from the 150 mg/kg/day dose group revealed no milk in stomach.
There were no interim death offspring in the 500 or 15 mg/kg/day groups.
No macroscopic abnormalities were detected at terminal kill.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Generation: F1, no adverse effects were observed up to the dose level of 500 mg/kg bw/d

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The oral administration of the substance to rats for a period of up to fifty-five consecutive days at dose levels of up to 500 mg/kg/day did not result in any treatment-related effects.
The ‘No Observed Effect Level’ (NOEL) was therefore considered to be 500 mg/kg/day.

Executive summary:

Introduction.

The study was perford to screen for potential adverse effects of the test material on reproduction including offspring developnt and provides an initial hazard assessnt for effect on reproduction. The study complies with the recomndations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developntal Toxicity Screening Test” (adopted).

This study was also designed to comply with Commission Regulation (EC) No 440/2008 of 30 May 2008 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 material was administered by gavage to three groups each of ten male and ten female Wistar Han™:HsdRccHan™:WIST strain rats, for up to fifty-five secutive days, (including a two week maturation phase, pairing, gestation and early lactation for females) at dose levels of 15, 150 and 500 mg//kg/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil).

Clinical signs, bodyweight developnt, 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 treatnt group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were perford on all surviving offspring, together with litter size and offspring weights and assessnt of surface righting reflex.

All adult males were terminated on Day 43, and all females and offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was perford.

Results.

Adult Responses:

Mortality.

There were no unscheduled deaths.

Clinical Observations.

No toxicologically significant clinical findings were observed throughout the study.

Bodyweight.

No adverse effect on bodyweight development was detected for treated animals, in comparison to controls.

Food Consumption and Food Efficiency.

No adverse effect on dietary intake was detected for treated animals, in comparison to controls.

Water Consumption.

There was no adverse effect on water consumption for treated animals, in comparison to controls.

Reproductive Performance:

Mating.

There were no treatment-related effects on mating rates.

Fertility.

There were no treatment-related effects on conception rates.

Gestation Length.

There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

Litter Responses:

Offspring Litter Size and Viability.

Of the litters born, litter size at birth and subsequently on Day 1 and 4 post partum the treated animals were comparable to controls.

Offspring Growth and Developnt.

Offspring bodyweight gain and litter weights at birth and subsequently on Day 1 and 4 post partum the treated animals were comparable to controls.

Pathology:

Necropsy.

The incidental signs detected in the adult and offspring were considered to be of no toxicological importance.

Uterine Examination.

From evaluation of the corpora lutea and implantation data, no adverse effects were detected for treated animals, in comparison to controls. 

Organ Weights.

No adverse effect on organ weight measurement was detected for treated animals, in comparison to controls.

Histopathology.

There were no treatment-related changes in the reproductive organs examined.

Conclusion.

The oral administration of ProductLE097 to rats for a period of up to fifty-five consecutive days at dose levels of up to 500 mg/kg/day did not result in any treatment-related effects.

The ‘No Observed Effect Level’ (NOEL) was therefore considered to be 500 mg/kg/day.