Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: OECD 422 Screening study
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Between 16 October 2009 and 08 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Wistar Han™:HsdRccHan™:WIST strain rat
Sex:
male/female
Details on test animals and environmental conditions:
Test Animals
- Source:
Wistar Han™:HsdRccHan™:WIST strain rats from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.

- Age at study initiation:
Approximately 12 weeks old

- Weight at study initiation:
297 to 342g (male); 184 to 233g (female)
- Fasting period before study:
Not applicable

- Housing:
Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). 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 within each dose group. 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:
The animals were allowed free access to food. A pelleted diet Rodent 2018C
Teklad Global Certified Diet Harlan UK Ltd, Oxon, UK was used throughout the study period. The diet was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

- Water:
Water intake was measured and recorded daily for each cage group (with the exception of non-recovery (satellite) animals during the mating phase). Individual daily water intakes were measures for females during the gestation and lactation phases of the study

- Acclimation period:
For 12 days

ENVIRONMENTAL CONDITIONS

- Temperature:
21 ± 2 °C

- Humidity:
55 ± 15 %

- Air changes (per hr):
At least fifteen air changes per hour

- Photoperiod (hr dark / hrs light):
12 hours continuous light and 12 hours darkness

IN-LIFE DATES:
20 October 2009 and 15 December 2009 (including recovery phase animals)

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: Not applicable
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test material formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services (Harlan Laboratories Ltd. Project Number: 0142-0416). Results from the previous study showed the formulations to be stable for at least twenty days. Formulations were therefore prepared twice monthly during the treatment period and stored at approximately +4ºC in the dark, under nitrogen.
Samples of each test material formulation were taken and analysed for concentration of test material at Harlan Laboratories Ltd., Shardlow, UK Analytical Services. The method used for analysis of formulations and the results obtained are given in Appendix 26. The results indicate that the prepared formulations were within plus or minus 9% of the nominal concentration.

DIET PREPARATION
- Not applicable

- Rate of preparation of diet (frequency):
Not applicable

- Mixing appropriate amounts with (Type of food):
Not applicable

- Storage temperature of food:
No data

VEHICLE
Arachis oil BP

- Justification for use and choice of vehicle (if other than water):
Not applicable

- Concentration in vehicle:
31.3, 7.5 and 2.5 mg/ml

- Amount of vehicle (if gavage):
4 ml/kg bodyweight

- Lot/batch no. (if required):
Not applicable

- Purity:
Not applicable
Details on mating procedure:

- M/F ratio per cage:
1/1 (Animals were paired on a 1 male: 1 female basis within each dose group)

- Length of cohabitation:
Up to 14 days

- 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 ... days of unsuccessful pairing replacement of first male by another male with proven fertility.:
Not applicable

- Further matings after two unsuccessful attempts:
Not applicable

- After successful mating each pregnant female was caged:
Mated females were housed individually during the period of gestation and lactation.

- Any other deviations from standard protocol:
Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9) in the test material formulations was determined by gas chromatography (GC) using an external standard technique.

The test material formulations were extracted with methanol to give a final, theoretical test material concentration of approximately 0.1 mg/ml. Procedural recoveries were performed at each dose level on every analysis occasion.

Standard solutions of test material were prepared in methanol at a nominal concentration of 0.1 mg/ml.

The analytical method has been satisfactorily validated in terms of specificity and accuracy for the purposes of the study.
Duration of treatment / exposure:
Non-recovery males from all treatment groups were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

(Following fourteen days without treatment, recovery control and high dose group males were terminated).
Frequency of treatment:
Daily
Details on study schedule:
Non-Recovery 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.

Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional / behavioural toxicity.

One day prior to pairing (Day 14), blood samples were taken from five males and five females, randomly selected from each dose group and analysed for haematological and blood chemical assessment.

On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of four 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.

On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli was performed.

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.

At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.

Additional blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically.

Additional blood samples were taken from five randomly selected females from each dose group at termination for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all surviving females and surviving offspring were killed and examined macroscopically.

The surviving high dose treatment animals were terminated early due to excessive toxicity. Additional blood samples were taken at termination for haematological and blood chemical assessments.

Recovery Dose Groups comprising of two groups (control and high dose) of five males were dosed for forty-two consecutive days. These males were then maintained without treatment for a further fourteen days.

Blood samples were taken for haematological and blood chemical assessment on Day 56. These animals were then killed and examined macroscopically.






Remarks:
Doses / Concentrations:
Dose levels of 10, 30 and 125 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
0 mg/kg/day – control: 10 animals per sex.
10 mg/kg/day : 10 animals per sex.
30 mg/kg/day : 10 animals per sex.
125 mg/kg/day : 10 animals per sex.
Recovery (Satellite ) 0 mg/kg/day – control: 5 males only.
Recovery (Satellite ) 125 mg/kg/day : 5 males only.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on Preliminary Fourteen Day Repeated Dose Oral (Gavage) Range-Finder in the Rat

- Rationale for animal assignment (if not random):
Random

- Rationale for selecting satellite groups:
To determine potential regression of any detected systemic responses elicited by administration of the test material

- Post-exposure recovery period in satellite groups:
Fourteen days

- Section schedule rationale (if not random):
Random
Positive control:
Not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:

- Yes see attached tables and appendices

- Time schedule:

- Immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, thirty minutes after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). During the treatment-free period, recovery males were observed once daily. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes (see above).
- Time schedule: As above.

NEUROBEHAVIOURAL EXAMINATION:

- Yes see attached tables and appendices

- Functional Observations were performed prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity.

- Functional performance tests (motor activity, forelimb/hindlimb grip strength and sensory reactivity) were also performed on five selected males during the final week of treatment and five Day 4 post partum females from each dose level.

BODY WEIGHT:

- Yes see attached tables and appendices

- Time schedule for examinations:

- Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating w as evident. Bodyweights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Bodyweights were
also recorded prior to termination

- For parameters checked see attached Tables.

FOOD CONSUMPTION:

- Yes see attached tables and appendices

- During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum. Weekly food consumptions were performed weekly for each cage of adults throughout the study period.

- FOOD EFFICIENCY:

- Yes see attached tables

- Food efficiency (the ratio of bodyweight change/dietary intake) was calculated retrospectively for males throughout the study period, and for females prior to mating.

WATER CONSUMPTION:

- Yes see attached tables

- Water intake was measured gravimetrically and recorded daily for each cage group (with the exception of non-recovery animals during the mating - phase). Individual daily water intakes were measured for females during the gestation and lactation phases of the study.

HAEMATOLOGY AND CLINICAL CHEMISTRY:

- Yes see attached tables and appendices

- Time schedule for collection of blood:

- Haematological and blood chemical investigations were performed on five males and five females selected from each non-recovery test and control group prior to termination (Day 42 for males and Day 4 post partum for females). These investigations were also performed on all recovery (satellite) males at the end of the treatment-free period (Day 56).

- Blood samples were obtained from the lateral tail vein or by cardiac puncture at termination, if applicable.

- Anaesthetic used for blood collection:
- No

- Animals fasted:
- No

URINALYSIS:
No

- Time schedule for collection of urine:
Not applicable

- Metabolism cages used for collection of urine:
Not applicable

- Animals fasted:
Not applicable

- Parameters examined:
Not applicable

OTHER:

MATING

- 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 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) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

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 mating
ii) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition
iv) Duration of gestation

LITTER SIZE

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

For each litter the following was recorded:

i) Number of offspring born
ii) Number and sex of offspring alive recorded daily and reported on Day 1 and 4 post partum
iii) Clinical condition of offspring from birth to Day 5 post partum
iv) Individual offspring weights on Day 1 and 4 post partum (litter weights were calculated retrospecively from offsring weights).

PHYSICAL DEVELOPMENT

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

- see attached tables and appendices

Oestrous cyclicity (parental animals):
A vaginal smear was prepared for each female and the stage of the oestrous cycle was recorded.
Sperm parameters (parental animals):
Parameters examined in all male parental generations:testis During histopathology, the male epididymides were examined for spermatocoel granuloma formation.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum:
No

PARAMETERS EXAMINED
The following parameters were examined in offspring:
Number of offspring born, number and sex of offspring alive recorded daily and reported on Day 1 and 4 post partum, clinical condition of offspring from birth to Day 5 post partum, individual offspring and litter weights on Day 1 and 4 post partum, physical Development and pathology.

GROSS EXAMINATION OF DEAD PUPS:
Dying offspring 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 surviving males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43.

- Maternal animals:
Adult surviving females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Any females that failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.

GROSS NECROPSY / ORGAN WEIGHTS

For all females the uterus was examined for signs of implantation and the number of uterine implantations in each born was recorded. This procedurewas enhanced; as necessary, by staining the uteri with a 1% ammonium polysulphide solution. In addition, the corpora lutea of all ovaries from pregnantfemales were counted at necropsy. All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY

The following organs, removed from the five selected males and parental females from each group that were killed at the end of the study, were dissected free from fat and weighed before fixation. Adrenals, ovaries, brain, spleen, epididymides, testes, heart, thymus, kidneys, thyroid and liver.

The following reproductive organs were weighed from all animals that were killed at the end of the study: ovaries, epididymides and testes.

Samples of the following tissues were preserved from five males and five females from each dose group, in buffered 10% formalin except where indicated.

Adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, coagulating gland, colon, duodenum, epididymides (preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) up to 48 hours later), eyes (fixed in Davidson’s fluid), gross lesions, heart, ileum, jejunum, kidneys, liver, lungs (with bronchi)(lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative), lymph nodes (cervical and mesenteric), mammarygland, muscle (skeletal), ovaries, pancreas, pituitary, prostate, oesophagus, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical), mid thoracic and lumbar, spleen, stomach, thyroid, trachea, testes (preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) up to 48 hours later), thymus, urinary bladder, uterus/cervix and vagina.

The following tissues were also removed from the remaining animals:coagulating gland, epididymides, ovaries, pituitary, prostate, seminal vesicles, testes and uterus/cervix.

All tissues were despatched to Harlan Laboratories Ltd, Switzerland (Principal Investigator: K Weber). The tissues from five selected control, 150 and 500 mg/kg/day dose group animals and those animals dying during the study, were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control, 150 and 500 mg/kg/day were also processed. Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of kidney and spleen from five animals per sex from the low dose groups.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.
Postmortem examinations (offspring):
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Necropsy findings checked in table 28 were included. All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
The volume of statistical references exceeds the storage capacity in this section it has therefore been included as an attachment titled 0142-0417 Statistics.
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 paired ÷ Number of animals mated) x 100
Pregnancy Index (%) = (Number of animals mated ÷ Number of pregnant females) 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 pregnant females ÷ Number of females delivering live offspring) x 100
Offspring viability indices:
The standard unit of assessment was considered to be the litter, therefore values were
first calculated for each litter and the group mean was calculated using their individual
litter values. Group mean values included all litters reared to termination (Day 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 Corpora Lutea) ÷ Number of implantation sites] x 100
% post – implantation loss =[(Number of implantation sites - Number of implantation sites) ÷ Total number of offspring born] x 100
ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring born ÷Number of offspring alive on Day 1) x 100
Viability Index 1 (%) = (Number of offspring alive on Day 1 ÷ Number of offspring alive on Day 4) 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
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422 1996)

RESULTS:

Mortality.

No unscheduled deaths were detected.

Clinical Observations.

A higher incidence of increased salivation was detected soon after dosing and up to one hour after dosing for animals of either sex treated with 125 and 30 mg/kg/day, and also for males treated with 10 mg/kg/day when compared to controls. Regression was evident following the cessation of treatment in recovery 125 mg/kg/day males.

Functional Observations.

No treatment-related effects were evident in the weekly behavioural assessments, sensory reactivity, grip strength or motor activity.

Bodyweight.

No adverse effect on bodyweight change was detected for males or for females during the pre-mating and gestation phases. Lower bodyweight gains were evident for females treated with 125 mg/kg/day when compared to controls during the lactation phase of the study. No adverse effects were evident at 30 or 10 mg/kg/day.

Food Consumption.

No adverse effects on dietary intake were evident for males or for females during the pre-mating or gestation phases of the study. A slight reduction in dietary intake was evident for females treated with 125 mg/kg/day when compared to controls during lactation.

Water Consumption.

No overt intergroup differences in water intake were detected for males or for females during the pre-mating or gestation phases of the study. A reduction in water intake was evident for females treated with 125 mg/kg/day when compared to controls during lactation.

Reproductive performance.

Mating.

No treatment-related effects were detected in mating performance.

Fertility.

No treatment-related effects were detected in fertility.

Gestation.

No treatment-related effects were detected on gestation length.

Litter responses.

Litter size and Viability.

Lower litter sizes, live birth indices and reduced numbers of viable litters were evident at 125 mg/kg/day when compared to controls. Slightly lower numbers in corpora lutea and implantation sites were evident for females treated with 125 mg/kg/day when compared to controls, and higher post-implantation losses were also evident.

Offspring Growth and Development.

Lower total litter weights were evident at 125 mg/kg/day in comparison to control values. Bodyweights and surface righting assessments were not
affected.

Laboratory Investigations.

Haematology.

Males treated with 125 mg/kg/day showed a reduction in haemoglobin, haematocrit, mean cell haemoglobin, mean cell volume and reticulocyte counts when compared to controls. These findings were considered to be of no toxicological significance.

No treatment-related effects were evident for females treated with 125 mg/kg/day, or for animals of either sex treated with 30 and 10 mg/kg/day.

Blood Chemistry.

No significant effects were detected in the blood chemical parameters investigated.

Pathology.

Organ Weights.

Males treated with 125 mg/kg/day showed slightly higher absolute and bodyweight-relative spleen and liver weights. These findings were considered to be of no toxicological importance.

No treatment-related effects were detected for females treated at 125 mg/kg/day, or for animals of either sex treated with 30 or 10 mg/kg/day.

Necropsy.

Offspring: No treatment-related macroscopic abnormalities were detected for offspring from treated animals when compared to control litters.

Adults: Treatment-related findings were confined to the presence of a thickened non-glandular region of the stomach for one male treated with
125 mg/kg/day.

Histopathology. The following treatment-related changes were observed:

STOMACH: Acanthosis, frequently with associated hyperkeratosis, was seen in the forestomach of all animals of either sex treated with 125 mg/kg/day, and in males treated with 30 mg/kg/day. There was evidence of regression of the condition in recovery 125 mg/kg/day males following an additional fourteen days without treatment.
Key result
Dose descriptor:
NOEL
Remarks:
Reproductive and development toxicity
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
other: Lower litter sizes due to lower numbers of corpora lutea and implantation sites, and higher post implantation losses were evident at 125 mg/kg/day. A NOEL was therefore considered to be 30 mg/kg/day for reproductive toxicity
Remarks on result:
other: Generation: Parents and offsprings (migrated information)
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
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
VIABILITY (OFFSPRING)

Lower numbers of corpora lutea and implantation sites were evident for females treated with 125 mg/kg/day when compared to controls, although statistical significance was achieved for lower numbers of implantation sites only, when compared to controls. A higher percentage of post-implantation losses was also evident at 125 mg/kg/day when compared to controls. This resulted in significantly lower litter sizes observed at birth for females treated with 125 mg/kg/day when compared to controls. Litter sizes on Day 1 and Day 4 of lactation were therefore also significantly smaller at 125 mg/kg/day when compared to controls. Lower live birth and viability indices were also evident at 125 mg/kg/day when compared to controls, although statistical analysis of the data did not reveal any significant intergroup differences.

Two litters from the 125 mg/kg/day dose group and three litters from the 30 mg/kg/day dose group showed dead offspring at birth. There were no offspring found dead at birth from the control or 10 mg/kg/day litters. A higher incidence of missing offspring (cannibalised by the mother following death) was evident at 125 mg/kg/day and possibly at 30 mg/kg/day in comparison to controls, although statistical analysis of this data did not reveal any significant intergroup differences.

The percentage of male offspring in the 125 mg/kg/day dose group was slightly lower than the number of male offspring observed in the control group, although statistical analysis was not achieved.

No treatment-related effects were evident for litters from the 10 mg/kg/day dose group.

A significantly lower number of implantation sites were noted at 10 mg/kg/day compared to controls (P<0.01). In isolation and in the absence of a dose-related response, this finding was not considered to be of any toxicological importance.

CLINICAL SIGNS (OFFSPRING)
No treatment-related clinical signs were detected. The clinical signs observed were low incidence findings commonly observed in reproductive studies of this type and unrelated to test material toxicity. Surface righting was not affected at any treatment level.

BODY WEIGHT (OFFSPRING)
Total litter weights were lower at 125 mg/kg/day on Day 1 (P<0.01) and Day 4 of lactation in comparison to control values. Bodyweights for offspring from treated animals were essentially similar to controls and no significant differences in bodyweight gains were evident between Day 1 and Day 4 of lactation.

SEXUAL MATURATION (OFFSPRING)
Not applicable

ORGAN WEIGHTS (OFFSPRING)
Not applicable

GROSS PATHOLOGY (OFFSPRING)
No treatment-related macroscopic abnormalities were detected for offspring dying during lactation or at termination on Day 5 post partum.

No treatment-related macroscopic abnormalities were detected at terminal kill.

The macroscopic abnormalities observed for interim death offspring consisted of autolytic changes, cannibalism, no milk present in the stomach and light brown colouration of the liver. Remaining macroscopic findings observed at termination were considered to be low incidence findings occasionally observed in reproductive studies of this type, and not related to test material toxicity.

HISTOPATHOLOGY (OFFSPRING)
Not applicable

OTHER FINDINGS (OFFSPRING)
Offspring Litter Size and Viability

Group mean corpora lutea and implantation counts, litter size, implantation losses, Lower numbers of corpora lutea and implantation sites were evident for females treated with 125 mg/kg/day when compared to controls, although statistical significance was achieved for lower numbers of implantation sites only, when compared to controls (P<0.05). A higher percentage of post-implantation losses was also evident at 125 mg/kg/day when compared to controls (P<0.01). This resulted in significantly lower litter sizes observed at birth for females treated with 125 mg/kg/day when compared to controls (P<0.001). Litter sizes on Day 1 and Day 4 of lactation were therefore also significantly smaller at 125 mg/kg/day when compared to controls (P<0.001). Lower live birth and viability indices were also evident at 125 mg/kg/day when compared to controls, although statistical analysis of the data did not reveal any significant intergroup differences.

Two litters from the 125 mg/kg/day dose group and three litters from the 30 mg/kg/day dose group showed dead offspring at birth. There were no offspring found dead at birth from the control or 10 mg/kg/day litters. A higher incidence of missing offspring (cannibalised by the mother following death) was evident at 125 mg/kg/day and possibly at 30 mg/kg/day in comparison to controls, although statistical analysis of this data did not reveal any significant intergroup differences.

The percentage of male offspring in the 125 mg/kg/day dose group was slightly lower than the number of male offspring observed in the control group, although statistical analysis was not achieved.

No treatment-related effects were evident for litters from the 10 mg/kg/day dose group.

A significantly lower number of implantation sites were noted at 10 mg/kg/day compared to controls (P<0.01). In isolation and in the absence of a dose-related response, this finding was not considered to be of any toxicological importance.


Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Reproductive effects observed:
not specified

See attached (0142-0417) Tables, Figures, Appendices, Addenda and Statistics.

 

The following results refer to the Fourteen day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat (Part 2) with Assessment of Maximum Tolerated Dose (Part 1)

Part 1: Maximum Tolerated Dose Results:

 

Mortality.

One 500 mg/kg/day female was found dead on Day 3 and the two remaining females were killedin extremison Day 3. There were no further unscheduled mortalities.

 

Clinical Observations.

No clinical observations were detected at 50 mg/kg/day and at 00 mg/kg/day findings were confined to one instance of transient increased salivation. At 200 mg/kg/day, increased salivation was detected in all animals. At 500 mg/kg/day, increased salivation was detected and accompanied on occasions by hunched posture and pilo-erection, tiptoe gait and ano-genital staining, lethargy and ptosis. All animals then showed a decline in general condition leading to the death of one animal and a decision was then taken on humane grounds to sacrifice the two surviving animals. Animals subsequently treated at 350 mg/kg/day displayed findings of increased salivation, diuresis, hunched posture, pilo-erection, decreased respiration, emaciation, ptosis, lethargy and pallor and diarrhoea.

 

Bodyweight.

Bodyweight gains were evident for females treated with 50 mg/kg/day. One female treated with 100 mg/kg/day showed a bodyweight loss of 1g on Day 3 although all females showed bodyweight gains on Day 5. One female treated with 200 mg/kg/day showed an 8g bodyweight loss on Day 3 and another female treated with 200 mg/kg/day showed a bodyweight loss of 5g on Day 5. Bodyweight gains at this dose level were less than those observed at 50 and 100 mg/kg/day. Bodyweight gains were evident for two females treated with 350 mg/kg/day on Day 3 and the bodyweight for one female was unchanged on Day 3 compared to the Day 1 bodyweight. Bodyweight losses of 3g and 29g were evident for two females treated with 350 mg/kg/day on Day 5. At 500 mg/kg/day, all females showed bodyweight losses of between 6 and 16g prior to treatment on Day 3, and further bodyweight losses of 1g and 6g were evident for the remaining two females prior to termination.

 

Necropsy.

The female treated with 500 mg/kg/day found dead on Day 3 showed a distended stomach, and sloughing of the glandular and non-glandular gastric epithelia. Gaseous distension was also observed in the small and large intestines. The remaining two females treated with 500 mg/kg/day and terminated on Day 3 showed gaseous distension of the gastro-intestinal tract. Females treated with 350 mg/kg/day were terminated following five days of treatment. One female (number 5) showed gaseous distension of the gastro-intestinal tract and sloughing of the non-glandular gastric epithelium. The remaining two females treated at this dose level did not reveal any macroscopic abnormalities.

 

Conclusion.

Oral administration of the test material to rats for up to five days at dose levels between 50 and 500 mg/kg/day resulted in significant toxicity at 500 and 350 mg/kg/day. The Maximum Tolerated Dose was therefore considered to be between 200 and 300 mg/kg/day.

 

Part 2: Fourteen day Repeated Dose Oral (Gavage) Range-Finding Toxicity StudyResults:

 

RESULTS

 

Mortality

Animals of either sex treated with 250 mg/kg/day were killedin extremison Day 10 following substantial bodyweight losses and a decline in physical health. There were no further unscheduled deaths.

 

Clinical Observations

One male treated with 250 mg/kg/day displayed increased salivation and noisy respiration soon after dosing from Day 1 and one female treated at this dose level displayed post-dose increased salivation from Day 2. Another female displayed diarrhoea on Days 3 and 4. Diarrhoea was also evident for one male on Day 3 and this male was observed as hunched on Day 4 and from Day 7 onwards. Incidents of increased salivation were also evident for remaining males from this dose group, from Day 2 and staining around the ano-genital region, suggestive of diarrhoea was observed in a number of animals of either sex from Day 4. On the morning of Day 10, clinical signs of lethargy, hunched posture, dehydration and diarrhoea were observed for all animals.

 

These clinical signs, together with bodyweight losses was considered excessive and the animals treated at this dose level were terminated on Day 10. Increased salivation was detected soon after dosing for animals of either sex treated with 150 mg/kg/day between Days 5 to 14. One male also displayed noisy respiration, although this was confined to Day 12 only.

 

No macroscopic abnormalities were detected for animals of either sex treated with

75 mg/kg/day.

 

Bodyweight

Substantial losses in bodyweight were evident for animals of either sex treated with 250 mg/kg/day, with the effect more prominent in males, which resulted in statistically significantly differences in this dose group when compared to control values (P<0.01).

 

These substantial bodyweight losses and the clinical signs observed, resulted in the

termination of this dose group on Day 10. Slight bodyweight losses were also evident for females treated with 150 mg/kg/day between Days 1 and 4 resulting in statistically significant reductions when compared to controls (P<0.05), although improvement was evident thereafter. The overall gain during the treatment period at 150 mg/kg/day was only slightly lower than controls (males -3.8%, females -2.9%), therefore, this was not considered to represent an adverse effect of treatment.

 

No adverse effect on bodyweight change was evident for animals of either sex treated with 75 mg/kg/day. Females treated with 75 mg/kg/day showed a statistically significant reduction in bodyweight gains (P<0.05), although this was only observed during the final four days of treatment.

 

Food Consumption

A reduction in dietary intake was evident for animals of either sex treated with 250 mg/kg/day prior to their early sacrifice on Day 10.

Slight reductions in dietary intake (approximately 11%) were also evident for animals of either sex treated with 150 mg/kg/day when compared to control values over the fourteen day treatment period. These reductions were considered not to represent an adverse effect of treatment.

 

No adverse effects on dietary intake were evident for animals of either sex treated with 75 mg/kg/day.

 

Water Consumption

Increases in water consumption were evident for animals of either sex treated with 250 mg/kg/day when compared to controls during the nine days of dosing, prior to their early sacrifice.

 

No adverse effects on water intake were evident for animals of either sex treated with 150 or 75 mg/kg/day.

 

Organ Weights

Animals of either sex treated with 150 mg/kg/day showed a statistically significant increase in absolute and relative liver weights when compared to controls (P<0.01).

 

The effect extended into the 75 mg/kg/day dose group, although statistical significance was only achieved for males (P<0.01).

 

Necropsy

 

The following macroscopic findings were observed for the high dose group terminated on Day 10: one male displayed pale lungs, a thickened urinary bladder with pale coloured contents, raised limiting ridge of the stomach, and a thickened and sloughing nonglandular region of the stomach. Another male showed reddened lungs and stomach changes consisting of gaseous distension, sloughing of the non-glandular region and a reddened appearance. The third male displayed a thickened non-glandular region which also showed sloughing. Sloughing of the non-glandular region was also evident for two females, one of which also showed a raised limiting ridge. One interim death female did not show any macroscopic abnormalities.

Two males and one female treated with 150 mg/kg/day displayed a thickened nonglandular region of the stomach, which was also evident for one female treated with 75 mg/kg/day.

 

No macroscopic abnormalities were detected for males treated with 75 mg/kg/day.

 

DISCUSSION

 

The oral administration of Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9) to rats by gavage for a period of up to fourteen consecutive days at dose levels of 250, 150 and 75 mg/kg/day resulted in treatment-related effects at all dose levels.

 

Clinical signs were observed at the highest dose level. These included increased salivation, noisy respiration, staining around the ano-genital region, diarrhoea and hunched posture. By Day 10, all animals showed lethargy, hunched posture, dehydration and diarrhoea. Significant bodyweight losses were also evident in this dose group, together with an increase in water intake and reduced dietary intake. Due to the severity of these effects, this dose level was considered excessive and the dose group was terminated on Day 10.Post-mortemexaminations revealed a number of effects, the most noticeable were stomach changes including raised limiting ridge, and thickened and sloughing of the gastric epithelia.

 

Clinical signs at 150 mg/kg/day were confined to isolated instances of increased salivation soon after dosing and noisy respiration. Slight bodyweight losses were evident for females during the first four days of treatment, although overall bodyweight change in this dose group was not adversely different from control values. There were no adverse effects on dietary intake detected at this dose level, althoughpost-mortemfindings revealed an increase in absolute and bodyweight-relative liver weights when compared to controls. Macroscopic examinations also revealed thickened non-glandular gastric epithelia for three animals from this treatment group.

 

Treatment-related effects at 75 mg/kg/day were confined to increases in absolute and bodyweight-relative liver weights, which were observed for animals of either sex, and a thickened non-glandular region of the stomach, which was observed for one female during thepost-mortemexaminations.

 

CONCLUSION

 

The oral administration of Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31- 9) to rats by gavage for a period of up to fourteen consecutive days at dose levels of 250, 150 and 75 mg/kg/day resulted in significant toxicity at 250 mg/kg/day.

 

Treatment-related effects including increased liver weights and macroscopic gastric changes were also evident at 150 and 75 mg/kg/day; therefore a ‘No Observed Effect Level’ (NOEL) was not established at the dose levels employed in the fourteen day range-finding phase.

 

 

 

Conclusions:
The oral administration of Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9) to rats by gavage, at dose levels of 125, 30 and 10 mg/kg/day, resulted in treatment related effects at 125 and 30 mg/kg/day. These effects consisted of a localised irritant effect, and almost complete regression was evident following the treatment-free period.

This change may be considered to be an adverse event and of importance when
considering the impact on reproductive performance. Based upon the histopathological changes observed in the stomach, a “No Observed Adverse Effect Level” (NOAEL) for systemic toxicity, was considered to be 30 mg/kg/day.

Lower litter sizes due to lower numbers of corpora lutea and implantation sites, and higher post implantation losses were evident at 125 mg/kg/day. A NOEL was therefore considered to be 30 mg/kg/day for reproductive toxicity.

In the study the controls showed a mean of 19.4 corpora lutea, which is outside the historical control range of 10-18. While the 125 mg/kg/day top dose females did show a reduced number of corpora lutea at a mean of 15.2 this was still well within the historical control range as was the number of implantations sites and there were no indications of a dose response. Due to the inevitable variability in the counting of corpora lutea in females four days after littering and the lack of a dose response these differences are not considered to be evidence of a toxic effect on reproduction.

The effects on the reproductive parameters were only seen in the 125 mg/kg bodyweight /day group, with no indication of a dose response at the lower doses. There was a combination of effects seen in the parental females in particular in the two litters which showed some of the most marked effects on the offspring survival which could be explained as possibly being secondary effects of maternal toxicity. The increase in post implantation loss was a more widespread phenomenon, however the marked increase was again influenced by females 71 and 72 which showed 9 and 5 post implantation losses and in addition female 77 which showed 11 post-implantation losses and produced no offspring. The remaining females in the 125 mg/kg bodyweight /day group while overall they still showed some increased post implantation loss individually the highest loss was 4 out of 14 which compared to one negative control female that showed 4 losses out of 17 implantation sites. As these effects on post implantation loss are concentrated particularly in three females it is possible that this is related to a toxic effect in those parental females rather than a more specific dose related development toxic effect in this group.

Due to the limitations of the study design of the OECD422 it is considered as a screening test for reproductive toxicity. It is not able to elucidate definitive reproductive effects particularly where they may involve developmental toxicity. Based on the findings in this study which indicate the possibility of foetal toxicity a full developmental toxicity study OECD 414 will be required to establish if these finding represent genuine developmental toxicity (foetal toxicity).
Executive summary:

ORAL (GAVAGE) COMBINED REPEAT DOSE TOXICITY STUDY WITH REPRODUCTION/DEVELOPMENTAL TOXICITY SCREENING TEST IN THE RAT (OECD 422 1996). PROJECT No. 0142-0417

  

Introduction.

The study was performed according to the protocol presented in Appendix 25 and was designed to screen for potential adverse effects of the test material on reproduction, including offspring development, following repeated oral administration to the Wistar Han™:HsdRccHan™:WIST strain rat for up to forty-five days(including a two week maturation phase, pairing, gestation and early lactation),at dose levels of 10, 30 and 125 mg/kg/day. A control group was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males, were treated with the high dose (125 mg/kg/day) or the vehicle alone for forty-two days and then maintained without treatment for a further fourteen days.

 

The study was designed to comply with the OECD Guidelines for Testing of Chemicals No. 422“Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).

 

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 forty-five consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 10, 30 and 125 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males, were treated with the high dose (125 mg/kg/day) or the vehicle alone for forty-two days and then maintained without treatment for a further fourteen days.

 

Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating and at termination on five selected males and females from each dose group. 

 

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 5 of lactation.

 

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

 

Extensive functional observations were performed on five selected males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4post partum.

 

Surviving males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5post partum.  All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Conclusion.

The oral administration of Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9) to rats by gavage, at dose levels of 125, 30 and 10 mg/kg/day, resulted in treatment-related effects at 125 and 30 mg/kg/day. These effects consisted of a localised irritant effect, and almost complete regression was evident following the treatment-free period. This change may be considered to be an adverse event and of importance when considering the impact on reproductive performance. Based upon the histopathological changes observed in the stomach, a “No Observed Adverse Effect Level” (NOAEL) for systemic toxicity, was considered to be 30 mg/kg/day.

 

Lower litter sizes due to lower numbers of corpora lutea and implantation sites, and higher post implantation losses were evident at 125 mg/kg/day.  A NOEL was therefore considered to be 30 mg/kg/day for reproductive toxicity.

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
125 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
High quality study but it is read across, awaiting final decision on testing proposal for a two generation reproduction study on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There is no specific test data on potential reproductive toxic effect of 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9. The available information is on a related substance Ethanol, 2,2’-iminobis-,N-coco alkyl derivs CAS No 61791-31-9, this substance is predominantly C12 but does contain >10% C18 while 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 is predominantly C18. The available study is an OECD 422 reproductive screening test. There were some indications of foetotoxicity in this study (post implantation loss) but this was considered not sufficient for classification, it was proposed to do additional testing to establish if there is developmental toxicity caused by the test substance . There were no indications of any effects on reproductive performance or fertility. The lack of potential for this is also supported by the lack of any adverse effects on the reproductive organs in the OECD408 90 day dosing study in rats on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9. There were no effects on the weight of the reproductive organs or seen when examined histopathologically. Also in addition the females were examined for any abnormality in the oestrus cycle and the males for abnormality in the spermatogenic cycle. No abnormalities were seen which supports the lack of potential for reproductive toxicity in the adult rats.

The read across to Ethanol, 2,2’-iminobis-,N-coco alkyl derivs CAS No 61791-31-9 is not ideal due to the difference in carbon chain length distribution. As 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 is a high volume substance >1000 tonnes we have proposed to do both a pre-natal developmental toxicity OECD 414 study (now completed) and a two generation reproduction study (delayed due to a disagreement between ECHA and some member states). The potential for reproductive toxicity in particular developmental toxicity (post implantation loss) with this substance will then be assessed based on the results of these studies which will be reviewed to see if any classification is required. 

Short description of key information:

There is no specific test data on potential reproductive toxic effect of 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9.  Therefore a test proposal has been made for a two generation reproduction study this is delayed awaiting a final decision from ECHA and the member states. The only available information is on a related substance Ethanol, 2,2’-iminobis-,N-coco alkyl derivs CAS No 61791-31-9, this substance is predominantly C12 but does contain >10% C18 while 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9  is predominantly C18.  The available study is an OECD 422 reproductive screening test.  There were no indications of adverse effects reproduction in the parental animals. There were some indications of foetotoxicity in this study but this was considered not sufficient for classification, it is proposed to do additional testing to establish if there is developmental toxicity (post implantation loss) also caused by 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9.

Justification for selection of Effect on fertility via oral route:

A two generation reproduction study has been proposed for this substance, until this is completed there is no specific test data on the potential adverse effects of 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 on fertility or other reproductive parameters.  The only available information is on a related substance Ethanol, 2,2’-iminobis-,N-coco alkyl derivs CAS No 61791-31-9, this substance is predominantly C12 but does contain >10% C18 while 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9  is predominantly C18.  The available study is an Klimisch 1, full GLP, OECD 422 reproductive screening test due to the shorter carbon chain length it is considered a worst case read across.

Justification for selection of Effect on fertility via inhalation route:

The low vapour pressure of the substance means inhalation is not considered to be relevant route of exposure so not testing is required.

Justification for selection of Effect on fertility via dermal route:

The corrosive properties of this substance mean the repeated dose dermal studies are not scientifically justified due to concerns for animal welfare.

Effects on developmental toxicity

Description of key information

There is an OECD414 pre-natal developmental toxicity study on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9. This study showed no indication of developmental toxicity in the foetuses, the NOEL being 150mg/kg.  There was however also no indication of toxicity in the pregnant females.  This study does not include dosing during the implantation period, the only available information concerning this is read across to the OECD422 study on Ethanol, 2,2’-iminobis-,N-coco alkyl derivs CAS No 61791-31-9.  This substance is predominantly C12 but does contain >10% C18 while 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9  is predominantly C18.  The available study is an OECD 422 reproductive screening test.  There were some indications of foetotoxicity (increased post implantation loss)  in this study but this was considered not sufficient for classification, it has been proposed to do a reproduction study to establish if this is also seen with 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19th June 2013 to 17 the December 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test”
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology Studies, 12 NohSan No 8147 (24 November 2000)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
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 delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 190g to 269g.

The animals were housed individually in solid-floor polypropylene cages with stainless steel 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, Harlan UK, Oxon, UK) was used. Certificates of analysis of the batches of diet used are given in Appendix 14. 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 Harlan Laboratories Ltd., 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 were 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.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
As Arachis Oil was successfully used on both the twenty-eight day and ninety toxicity studies, the same vehicle and dosage (4 mL/kg body weight) was employed in this study. The stability and homogeneity of the test item formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services and showed the formulations to be stable for at least twenty one days at 4 °C. Formulations were therefore prepared in two separate bulk formulations (covering up to 9 days) and divided into daily aliquots and stored at approximately +4 °C in the dark.

Samples were taken of each test item formulation and were analyzed for concentration of 2,2'-(octadec-9-enylimino)bisethanol CAS No 25307-17-9 at Harlan Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within 94% to 104% of the nominal concentration and within acceptable limits of the nominal concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

Test Item
The test item described in the main part of this study was also used as the analytical standard.

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

Analysis of samples
The formulations recieved were extracted with methanol. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with methanol. This was then ultra-sonicated for 15 minutes and centrifuged to 4500 rpm for 10 minutes. Where necessary, sample solutions were further diluted with methanol to achieve the working concentration.

Preparation of accuracy samples
Samples of Arachis Oil BP were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These sample were then prepared for analysis.

Instrumental Setip

HC system: Agilent Technologues 5890, incorporating autosampler and workstation
Column: DB-1 (15 m x 0.53 mm id x 1.5 micro-m film)
Oven temperature program: Oven: 200°C for 0 minute, with 10°C/minute to 300°C, for 12 minutes
Injection temperature: 300°C
Flame ionisation detector temperature: 300°C
Injection volume: 1 micro-litre
Retention time: ~ 4.5 mins


Details on mating procedure:
Not described in the study
Duration of treatment / exposure:
Between Days 5 and 19 of gestation, inclusive.
Frequency of treatment:
Daily
Duration of test:
20 days
Remarks:
Doses / Concentrations:
15 mg/kg/day (3.75 mg/ml)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg/day (12.5 mg/ml)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg/day (37.5 mg/ml)
Basis:
actual ingested
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
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.

Justification
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Maternal examinations:
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. An additional observation was also performed five hours after dosing during the normal working week. 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:
Post Mortem
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

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 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
Fetal examinations:
The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed 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 anomalies and storage.
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: Bartlett’s test for homogeneity of variance. Where the data were shown to be homogeneous one way analysis of variance and, if significant, Dunnett’s multiple comparison test was employed, where the data were found to non homogeneous Kruskal-Wallis and, if significant, pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test was employed. Fetal evaluation parameters, including skeletal or visceral findings were analyzed by Kruskal-Wallis and, if significant, Mann-Whitney ‘U’ test.

Probability values (p) are presented as follows:

p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No effects of any toxicological significance
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter Responses
Litter Data and Litter Placental and Fetal Weights
There was no obvious effect of maternal treatment on the number of implantations, subsequent embryofetal survival and litter size, sex ratio and mean fetal, litter and placental weights on Day 20 of gestation at 15, 50 or 150 mg/kg bw/day.

At 150 mg/kg bw/day, mean pre-implantation loss was lower than control with differences attaining statistical significance. As animals were not dosed until implantation had occurred, these differences were incidental and unrelated to treatment.

At 15 and 50 mg/kg bw/day, higher mean female fetal weight and mean fetal weight attained statistical significance compared to control. In the absence of any similar increase in fetal weight at 150 mg/kg bw/day, this finding was considered to reflect normal biological variation and was unrelated to treatment.

Fetal Examination
Neither the type, incidence or distribution of findings observed externally at necropsy examination and subsequently during detailed visceral and skeletal assessment of the fetuses indicated any effect of treatment on fetal development.
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Mortality There were no unscheduled deaths during the study. Clinical Observations The low incidence of clinical sign observed during the study did not indicate any effect of treatment at 15, 50 or 150 mg/kg bw/day. Body Weight There was no effect of treatment on body weight or body weight gain, including when adjusted for the contribution of the gravid uterus, throughout the treatment period at 15, 50 or 150 mg/kg bw/day. Food Consumption There was no effect of treatment on food consumption throughout the treatment period at 15, 50 or 150 mg/kg bw/day. Water Consumption Daily visual inspection of water bottles did not reveal any overt intergroup differences. Post Mortem Studies No macroscopic abnormalities were detected for parental females at scheduled termination on Day 20 of gestation. Litter Responses Litter Data and Litter Placental and Fetal Weights There was no obvious effect of maternal treatment on the number of implantations, subsequent embryofetal survival and litter size, sex ratio and mean fetal, litter and placental weights on Day 20 of gestation at 15, 50 or 150 mg/kg bw/day. At 150 mg/kg bw/day, mean pre-implantation loss was lower than control with differences attaining statistical significance. As animals were not dosed until implantation had occurred, these differences were incidental and unrelated to treatment. At 15 and 50 mg/kg bw/day, higher mean female fetal weight and mean fetal weight attained statistical significance compared to control. In the absence of any similar increase in fetal weight at 150 mg/kg bw/day, this finding was considered to reflect normal biological variation and was unrelated to treatment. Fetal Examination Neither the type, incidence or distribution of findings observed externally at necropsy examination and subsequently during detailed visceral and skeletal assessment of the fetuses indicated any effect of treatment on fetal development.
Conclusions:
The No Observed Effect Level (NOEL) for the pregnant females and the survival, growth and embryofetal development of the offspring was considered to be 150 mg/kg bw/day.
Executive summary:

Introduction

The study was designed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.

 

The study was designed to comply with the following guidelines:

 

·        US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

·        Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

·        OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

·        Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (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 twenty-four time mated Sprague-Dawley Crl:CD®(SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 15, 50, and 150 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Arachis oil) over the same treatment period 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….

Adult Responses

Mortality

There were no unscheduled deaths during the study.

 

 Clinical Observations

Clinical sign did not indicate any effect of treatment at 15, 50 or 150 mg/kg bw/day.

  

Body Weight

Body weight and body weight gain, including adjustment for the contribution of the gravid uterus, was unaffected by treatment at 15, 50 or 150 mg/kg bw/day.

 

 Food Consumption

Food consumption was unaffected by treatment at 15, 50 or 150 mg/kg bw/day.

 

 Water Consumption

Daily visual inspection of water bottles did not reveal any overt intergroup differences.

 

 Post Mortem Studies

No macroscopic abnormalities were detected for parental females at 15, 50 or 150 mg/kg bw/day.

 

 Litter Responses

Litter Data and Litter Placental and Fetal Weights

The number of implantations, subsequent embryofetal survival and litter size, sex ratio and mean fetal, litter and placental weights on Day 20 of gestation were unaffected by maternal treatment at 15, 50 or 150 mg/kg bw/day.

 

 Fetal Examination

There was no effect of maternal treatment on morphological development of the fetuses at 15, 50 or 150 mg/kg bw/day.

 

 Conclusion

The No Observed Effect Level (NOEL) for the pregnant females and the survival, growth and embryofetal development of the offspring was considered to be 150 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
This study is high quality and replaces the read across to Ethanol, 2,2’-iminobis-,N-coco alkyl derivs CAS No 61791-31-9 for developmental toxicity but it does not cover the period immediately after implantation so it does not supersede the read across to the OECD422 study where increased post implantation loss was seen. This will be resolved when the proposed reproduction study on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 is available.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The OECD414 pre-natal development study included dose levels of 15, 50 and 150mg/kg bodyweight of 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9, administered by gavage for days 5 to 19 of pregnancy as a solution in arachis oil. The dose levels were selected based on those used in the 28 day and 90 day repeat dose studies. The adult females did not show any signs of toxicity in this study such as on bodyweights, or food or water consumption, but there was no histopathological examination of their stomachs making it not possible to see local adverse (irritant/corrosive) effects due to the corrosive/irritant nature of the test material as seen in the other repeat dose studies.

The absence of toxic effects in the adult females is not considered to be of concern as we have good evidence from the 28 day repeat dose study that higher doses could result in mortality or severe toxicity due to local effects in the stomach. 

The number of implantations, subsequent embryofoetal survival and litter size, sex ratio

and mean foetal, litter and placental weights on Day 20 of gestation were unaffected by maternal treatment at 15, 50 or 150 mg/kg bw/day.

 

There was no effect of maternal treatment on morphological development of the foetuses at 15, 50 or 150 mg/kg bw/day.

 

Conclusion

 

The No Observed Effect Level (NOEL) for the pregnant females and the survival, growth and embryofoetal development of the offspring was considered to be 150 mg/kg bw/day.2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9, did not show any indications of potential for developmental toxicity in this study. The proposed reproduction study will allow an assessment if the increased post implantation loss seen with the read across substance 2,2’-iminobis-,N-coco alkyl derivs CAS No 61791-31-9 is also seen with this substance.

 

Justification for selection of Effect on developmental toxicity: via oral route:

We have a Klimisch 1 full GLP compliant OECD414 study on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9.

Justification for selection of Effect on developmental toxicity: via inhalation route:

The low vapour pressure of the substance means inhalation is not considered to be relevant route of exposure so not testing is required.

Justification for selection of Effect on developmental toxicity: via dermal route:

The corrosive properties of this substance mean the repeated dose dermal studies are not scientifically justified due to concerns for animal welfare.

Justification for classification or non-classification

There is no specific test data on potential reproductive toxic effect of 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9. The available information is on a related substance Ethanol, 2,2’-iminobis-,N-coco alkyl derivs CAS No 61791-31-9, this substance is predominantly C12 but does contain >10% C18 while 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 is predominantly C18. The available study is an OECD 422 reproductive screening test. There were some indications of foetotoxicity (increased post implantation loss) in this study but this was considered not sufficient for classification, it was proposed to do additional testing to establish if there is true developmental toxicity. There were no indications of any adverse effects on the reproductive parameters such as fertility in the adult rats of either sex. The lack of potential for this is also supported by the lack of any adverse effects on the reproductive organs in the OECD408 90 day dosing study in rats on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9. There were no effects on the weight of the organs or when examined histopathologically. Also in addition the females were examined for any abnormality in the oestrus cycle and the males for abnormality in the spermatogenic cycle. No abnormalities were seen which supports the lack of potential for reproductive toxicity.

The read across to Ethanol, 2, 2’-iminobis-, N-coco alkyl derivs CAS No 61791-31-9 is not ideal due to the difference in carbon chain length distribution. As 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 is a high volume substance >1000 tonnes we have proposed to do both a developmental toxicity OECD 414 study (which is now completed) and a two generation reproduction study which is currently awaiting a final decision due to differences of opinion between some member states and ECHA on the replacement of the two generation study with an Extended one generation study.

 

The OECD414 pre-natal development study in rats, showed no evidence of developmental toxicity even at the top dose of 150mg/kg bodyweight therefore while this study does not include dosing during the pre-mating period until day 4, it does not indicate any potential for developmental toxicity. Until the reproduction study is available it is not possible to know if the increased post implantation loss see in the OECD422 study with the read across substance Ethanol, 2,2’-iminobis-,N-coco alkyl derivs CAS No 61791-31-9 will also be seen with this substance. However the evidence from the OECD414 study indicates that 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 does not induce developmental toxicity in rats, therefore it is clear that it is not necessary to classify this substance for reproductive or developmental toxicity based on the current data. A final conclusion concerning the post implantation loss and its relevance to this substance will be possible when the reproduction study is completed.