Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The is an OECD422 reproduction screening study on the test substance which did not show any adverse effects on reproduction with no effects on fertility, mating, gestation period. There were no effects on any of the reproductive organs.

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: OECD422 Reproductive screening test
Type of information:
experimental study
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
Description (incidence and severity):
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 treat ment in recovery 125 mg/kg/day males
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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 and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
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.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Clinical biochemistry findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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.
Histopathological findings: neoplastic:
no effects observed
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:
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)
Critical effects observed:
yes
Lowest effective dose / conc.:
125 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
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.
Body weight and weight changes:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
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 they still showed some increased post implantation loss individually, the highest loss was of 4 out of 14 implantations, which compared to one negative control female that showed 4 losses out of 17 implantations. 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
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Bis(2-hydroxyethyl) coco alkylamine CAS No 61791-31-9 has been tested in the OECD422 test which is a screening study for reproductive toxicity. There were no indications in this study of any adverse effect on mating performance, gestation length or fertility with dose levels up to 125 mg/kg/day.

Short description of key information:

Bis(2-hydroxyethyl) coco alkylamine CAS No 61791-31-9 will be registered for REACH as

2,2’-(C12-18 even numbered alkyl imino) diethanol CAS No71786-60-2. This substance has been tested in the OECD422 test which is a screening study for reproductive toxicity. There were no indication in this study of any adverse effect on fertility with dose levels up to 125 mg/kg/day.

Justification for selection of Effect on fertility via oral route:

This is a full GLP Klimisch 1 OECD422 reproduction screening study carried out with the substance to be registered 2, 2’-(C12-18 evennumbered alkyl imino) diethanol, CAS No 71786-60-2, described at the time of testing as Ethanol, 2, 2’-iminobis-, N-coco alkyl derives CAS No 61791-31-9, these are the same substance

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

Bis(2-hydroxyethyl) coco alkylamine CAS No 61791-31-9 will be registered for REACH as

2,2’-(C12-18 even numbered alkyl imino) diethanol CAS No71786-60-2. This substance has been tested in the OECD422 test which is a screening study for reproductive toxicity. In this study there were some indications in the top dose of 125 mg/kg/day of possible developmental toxicity, in particular post-implantation loss indicating possible foetotoxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
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: 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:
other: OECD Guidelines for Testing of Chemicals No. 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:
Wistar
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 Laboratories U.K. 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:
Up to 45 consecutive days for females and 42 days for males.



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 +/- 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
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.

See attached Appendix 26 - Chemical Analysis of Test Material Formulations, Methods
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
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
Duration of test:
The in-life phase of the study was conducted between 20 October 2009 (first day of treatment) and 15 December 2009 (final necropsy).
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-Finding Toxicity Study in the Rat (0142-0416)

- 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
Maternal 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 offspring weights).

PHYSICAL DEVELOPMENT

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

- see attached tables and appendices

Ovaries and uterine content:
The ovaries and uterine content was examined after termination:
Yes
Examinations included:
- Gravid uterus weight:
No

- Number of corpora lutea:
Yes

- Number of implantations:
Yes

- Number of early resorptions:
No

- Number of late resorptions:
No

- Other:
Parameters in Table 1 were examined in this study.
Fetal examinations:
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 weights on Day 1 and 4 post partum, physical Development and pathology.

GROSS EXAMINATION OF DEAD PUPS:
Any dead and dying offspring that may have occurred during the study were subjected to a full external and internal examination, and any macroscopic abnormalities recorded.

POST-MORTEM EXAMINATION
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Necropsy findings checked in table 21 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.
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
Historical control data:
Included: see Addenum 3-7
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
A higher incidence of increased salivation was detected soon after dosing and up to one hour after dosing for animals of treated with 125 mg/kg/day and 30 mg/kg/day.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant reduction in bodyweight gains was observed for post partum females treated with 125 mg/kg/day when compared to controls during the early lactation.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant increase in absolute and bodyweight-relative liver weights was observed for males treated with 125 mg/kg/day. Males treated with 125 and 30 mg/kg/day showed slight but statistically significant increases in absolute and bodyweight-relative thyroid weights (P<0.05) when compared to controls.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were confined to the presence of a thickened glandular and non-glandular region of the stomach recorded for one non-recovery male treated with 125 mg/kg/day at the end of the treatment period.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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 only 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, with just two animals affected.
Histopathological findings: neoplastic:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
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.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, treatment-related
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
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.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Adult Responses
Mortality
No unscheduled deaths were detected during the study.

Clinical Observations
A summary incidence of clinical observations is given in Table 2. Individual clinical observations are presented in Appendix 1.
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 mg/kg/day, with the effect also evident for animals of either sex
treated with 30 mg/kg/day, albeit at a lesser extent and this was also evident for males treated with 10 mg/kg/day when compared to controls. Isolated incidents of noisy respiration and staining around the mouth
were also noted for males treated with 125 mg/kg/day. Regression was evident following the cessation of treatment in recovery 125 mg/kg/day males.
Remaining clinical observations were confined to fur loss observed for one male treated with 125 mg/kg/day between Days 2 to 8 and for one female treated with 10 mg/kg/day on Day 42. This finding is occasionally observed in laboratory maintained animals, and is considered to be unrelated to treatment.

Functional Observations
A summary incidence of behavioural assessment observations is given in Table 3 and group mean behavioural assessment scores are given in Table 4. Group mean functional test values and standard deviations are
given in Table 5. Individual values are given in Appendix 2 and Appendix 3. Group mean sensory reactivity assessment scores are given in Table 6. Individual responses are given in Appendix 4.

Behavioural Assessments: Weekly open-field arena observations did not reveal any treatment-related effects detected for treated animals when compared to controls.
All inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used, and the differences were of no toxicological importance.

Functional Performance Tests: No treatment-related effects were evident in grip strength or motor activity for treated animals when compared to controls.
Statistical analysis of the data did not reveal any significant intergroup differences.

Sensory Reactivity Assessments: No treatment-related effects were evident in sensory reactivity scores for treated animals when compared to controls.
All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and the differences were of no toxicological importance.

Bodyweight
Group mean bodyweights and standard deviations are given in Table 7 and are presented graphically in Figure 1 and Figure 2. Group mean bodyweight gains and standard deviations are given in Table 8 (statistically significant differences are indicated). Individual data are given in Appendix 5 and Appendix 6.
No adverse effect on bodyweight change was detected for treated males during the treatment or recovery phases of the study when compared to controls.
No adverse effects on bodyweight change were detected for females during the pre-mating or gestation phases of the study. A statistically significant reduction in bodyweight gains was observed for post partum females treated with 125 mg/kg/day when compared to controls during the early lactation (P<0.01).
No adverse effects were evident for females treated with 30 or 10 mg/kg/day.

Food Consumption
Group mean food consumptions are given in Table 9 and are presented graphically in Figure 3 and Figure 4. Weekly food efficiencies for males, and for females during the pre-mating phase, are given in Table 10. Individual and group mean food consumptions for females following mating and during lactation are presented in Appendix 7.
No adverse effects on dietary intake or food conversion efficiency (the ratio of bodyweight gain/dietary intake) were evident for males during the treatment or recovery phases of the study.
No adverse effects on dietary intake were evident for females during the pre-mating and 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. Statistical analysis of the data for females during gestation and lactation however did not reveal any significant intergroup differences.
Food conversion efficiency during the pre-mating phase was not affected.

Water Consumption
Group mean daily water consumptions are given in Table 11. Individual and group mean daily water consumptions for females following mating and during lactation are presented in Appendix 8.
No overt intergroup differences in water intake were detected for males during the treatment or recovery phases of the study.
No adverse effects on water intake were observed for females during the pre-mating or gestation phases of the study. Statistically significant reductions in water intake were observed for females treated with 125 mg/kg/day when compared to controls during the early lactation phase (P<0.01 – P<0.001).

Reproductive Performance

Mating
A summary of adult performance is given in Table 1. Group values for mating performance are presented in Table 12. Individual data are given in Appendix 9.
No treatment-related effects were detected in mating performance. All paired animals from all treatment and control groups mated within the first four days of pairing.

Fertility
A summary of adult performance is given in Table 1. Group values for fertility, litter data and implantation losses are given in Tables 12, 13 and 14. Individual data are given in Appendices 9 to 11.
No treatment-related effects were detected in fertility between control and treated animals.
One female treated with 10 mg/kg/day did not achieve pregnancy following confirmation of mating. This was an isolated finding occasionally observed in reproductive studies of this type, and is not considered to represent an effect of treatment.

Gestation Length
A summary incidence of gestation lengths are given in Table 12. Individual lengths are given in Appendix 9.
No treatment-related effects were detected in the length of gestation for treated pregnant females when compared to controls.
Statistical analysis of the data did not reveal any significant intergroup differences.

Litter Response
In total, all paired animals mated, and all mated females from the intermediate and control groups gave birth to a live litter and successfully reared young to Day 5 of age. There was one non-pregnant female in the low dose group (10 mg/kg/day), and one female treated at the high dose group mated and showed corpora lutea and implantation sites but did not produce offspring. This was considered to be a total litter loss in utero. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size and Viability
Group mean corpora lutea and implantation counts, litter size, implantation losses, survival indices and sex ratio are given in Tables 13 to 15 (statistically significant differences are indicated). Individual data are given in Appendices 10 to 12.
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 (cannabalised 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.

Offspring Growth and Development
Group values for offspring bodyweight and bodyweight change, surface righting reflex and the incidence of clinical signs are given in Tables 13, 16 and 17 (statistically significant differences are indicated). Individual values and observations are given in Appendices 10, 13 and 14.
Total litter weights were lower at 125 mg/kg/day on Day 1 (P<0.01) and Day 4 of lactation (P<0.001) 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.
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.

Laboratory Investigations

Haematology
Group mean values and standard deviations for test and control group animals are given in Table 18 (statistically significant differences are indicated). Individual data are given in Appendix 15 to Appendix 18.
Males treated with 125 mg/kg/day showed statistically significant reductions in haemoglobin and mean cell volume (MCV) levels when compared to controls (P<0.01). Slight reductions in haematocrit, mean cell haemoglobin (MCH) and reticulocyte counts were also evident at this dose level in comparison to controls (P<0.05). Reduced MCV and MCH levels were still evident for recovery males following the fourteen day treatment-free period.
No treatment-related effects were observed for females treated with 125 mg/kg/day, or for animals of either sex treated with 30 and 10 mg/kg/day.
MCV was also lower for males treated with 30 and 10 mg/kg/day in comparison to the control values (P<0.01). All individual values were within the normally expected ranges and in the absence of any further changes in the spleen at these dose levels, and the absence of a dose-related response, these findings were considered to be of no toxicological importance.
An increase in platelet counts was evident for males treated with 125 mg/kg/day when compared to controls at the end of the treatment period. The significance achieved was minimal (P<0.05) and all individual values from the treated group were within the normally expected ranges for this parameter. This increase was considered to be attributable to one lower than expected control value, and was therefore unrelated to treatment with the test material.

Blood Chemistry
Group mean values and standard deviations for test and control group animals are given in Table 19 (statistically significant differences are indicated). Individual data are given in Appendix 19.
No toxicologically significant effects were detected in the blood chemical parameters investigated.
Post partum females treated with 125 mg/kg/day showed a statistically significant reduction in bilirubin when compared to controls. The significance achieved was minimal (P<0.05) and all individual values for control and high dose animals were within the normally expected ranges for this parameter. There were no further blood chemical changes observed at this dose level. Therefore, this minor reduction was not considered to be of any toxicological importance. Post partum females treated with 30 mg/kg/day also showed a slight but statistically significant increase in alkaline phosphatase levels when compared to controls (P<0.05). Two individual values were outside the normally expected ranges. An increase in alkaline phosphatase is commonly observed in pregnancy due to production by the placenta, and this slight increase may be due to this. Furthermore, a dose-related response was not observed and in the absence of any associated histopathological correlates, this increase was of no toxicological importance.
Recovery 125 mg/kg/day males showed a slight but statistically significant increase in total protein levels when compared to their concurrent controls. The significance achieved was minimal (P<0.05) and all individual values were within the normally expected ranges for this parameter. In isolation, this increase was considered to have arisen incidentally.

Pathology

Organ Weights
Group mean absolute and relative organ weights and standard deviations for test and control group animals are presented in Table 20 (statistically significant differences are indicated). Individual data are given in Appendix 20 and Appendix 21.
A statistically significant increase in absolute and bodyweight-relative liver weights was observed for males treated with 125 mg/kg/day. Although one low control value was observed for absolute weights, three absolute weight values and four bodyweight-relative weight values were outside of the respective historical ranges in the 125 mg/kg/day dose group.
Absolute and bodyweight-relative spleen weights were elevated for males treated with 125 mg/kg/day at the end of the treatment period, and this was still evident for recovery 125 mg/kg/day males following the fourteen day treatment free period, when compared to controls (P<0.05). All values were within the normally expected ranges for this parameter.
There were no further organ weight changes which were considered to be attributable to the administration of the test material in the male dose groups. No treatment-related organ weights were evident for females at all dose levels.
Males treated with 125 and 30 mg/kg/day showed slight but statistically significant increases in absolute and bodyweight-relative thyroid weights (P<0.05) when compared to controls. All individual values at the highest dose level were within the normally expected ranges for these parameters, and only one absolute thyroid weight value was lower than the expected range at 30 mg/kg/day. Two lower than expected values in both the absolute and bodyweight-relative thyroid weights were observed in the control groups. In the absence of a convincing dose-related response and in the absence of a treatment-related effect following histopathological examinations of the thyroids, the slight increases were considered to be attributed to the lower than expected values observed in the control groups, and were of no toxicological importance. Finally, slight but statistically significant increases in absolute and bodyweight-relative kidney weights were observed for males from all dose levels when compared to controls. The statistical significance achieved in each occasion was P<0.05. All individual values at 125 and 30 mg/kg/day were within the normally expected ranges. With the exception of one absolute kidney weight, all individual values at the lowest dose level were also within the normally expected limits. One lower than expected absolute kidney weight value and two lower than expected bodyweight-relative kidney weight values were observed for the controls. In the absence of a convincing dose-related response and the absence of microscopic changes observed in the kidneys following histopathological assessments, these slight increases were considered to be attributed to the lower than expected control values, and were of no toxicological significance.

Necropsy
A summary incidence of necropsy findings is given in Table 21 and Table 22. Individual data are given in Appendices 22 and 23.
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.
Adults
Treatment-related findings were confined to the presence of a thickened glandular and non-glandular region of the stomach recorded for one non-recovery male treated with 125 mg/kg/day at the end of the treatment period.
There were no further macroscopic abnormalities which were considered to be attributable to treatment with the test material.
A reddened median lobe of the liver was evident for one male treated with 30 mg/kg/day. Sloughing of the glandular gastric epithelia was observed for one female treated with 10 mg/kg/day, although sloughing of the glandular and non-glandular epithelia was also recorded for one control female. In the absence of any histopathological changes in the stomach observed at this dose level, the toxicological importance of this finding was minimal.

Histopathology
A summary incidence of histopathological findings is given in Table 23. Individual data are given in Appendix 24.
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 only 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, with just two animals affected.
Remaining histopathological changes seen among surviving control and high dose animals were all considered to be spontaneous in origin and unrelated to treatment. The following conditions warrant specific mention:
ADRENAL GLANDS: Cortical vacuolation was seen in a few control and treated males and was of no toxicological significance in this investigation.
BONE MARROW: Adipose infiltration of the marrow is an indicator of changes in marrow cellularity and in this study, there was no difference between control and treated groups.
KIDNEYS: Focal corticomedullary mineralisation is a commonly observed background condition among females. Globular accumulations of eosinophilic material, as a consequence of excessive accumulation of alpha2-microglobulin in renal proximal tubular epithelial cells, are occasionally encountered as a spontaneous change in male rats.
LIVER: Scattered mononuclear cell foci were observed in a few control and treated animals examined in the study. Such are commonly observed in the rodent liver and are not indicative of any adverse condition at the severities encountered.
LUNGS: A minimal severity of bronchus associated lymphoid tissue was reported for all control and high dose animals examined in the study and is not indicative of respiratory disease. Minor severities and low incidences of focal pneumonitis and accumulations of alveolar macrophages are commonly observed pulmonary changes in laboratory maintained rats of this age and are not suggestive of significant respiratory disease.
SPLEEN: Extramedullary haemopoiesis is a normal background condition in the rat spleen and the severities observed were considered to be within normal limits.
THYROID: Follicular cell hypertrophy is commonly seen among untreated animals of either sex and there was no indication of a relationship to treatment in this study.
THYMUS: Lymphoid atrophy is frequently seen among pregnant and lactating female rats.
REPRODUCTIVE TRACT AND RELATED ORGANS
PITUITARY: No treatment-related changes were seen. Vacuolation of pars anterior cells is a commonly observed change more especially in male rats.
TESTIS/EPIDIDYMIS: Testicular atrophy is observed occasionally as a spontaneous condition frequently with associated cellular debris in the epididymis.
SEMINAL VESICLES/COAGULATING GLAND: No treatment-related changes were seen.
PROSTATE: No pathological changes were seen.
MAMMARY GLAND: Glandular hyperplasia was observed in the mammary tissue of all previously pregnant females and is consistent with pregnancy and lactation.
OVARY: No pathological changes were seen.
UTERUS/VAGINA: Areas of haemorrhage and fibrosis were seen in the myometrium and adjacent connective tissue of the uterus in the majority of females examined from control and high dose groups. These conditions are consistent with normal post partum uterine changes in the rat. Keratinisation of the uterine cervix and/or vagina is a normal cyclical change in female rats.
All other morphological changes in the above and remaining tissues 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.

Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: other:
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day
Basis for effect level:
other: other:
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
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.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
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.
Changes in sex ratio:
effects observed, non-treatment-related
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The following assessment of litter response is generally based on those litters reared to termination on Day 5 post partum, although data available for females showing total litter loss has also been taken into consideration, where considered appropriate.

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.

Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
Abnormalities:
not specified
Developmental 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) (Harlan Laboratories Ltd., Project Number 0142 -0416)

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 Study Results:

 

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 they still showed some increased post implantation loss individually, the highest loss was of 4 out of 14 implantations, which compared to one negative control female that showed 4 losses out of 17 implantations. 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 4 post partum.

 

Surviving males 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.

 

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.

 

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27th March 2018 -12th June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Test item: Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives
Test item identity (including alternative names):
Ethomeen C/12
2,2’-(C12-18 even numbered alkyl imino)diethanol.
Bis(2-hydroxyethyl)cocoalkylamine.
CAS number: 71786-60-2 and 61791-31-9
Intended use: Substance used in industry.
Appearance: Light yellow liquid.
Storage conditions: At ambient temperature (15 to 25C), in the dark.
Supplier: Sponsor
Batch number: 1373142
Stability/expiry date: 28 November 2018
Purity: 98%
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Strain/Species RccHan™:WIST rat.
Supplier Envigo (RMS) UK Limited
Number of animals ordered 90 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization Five days before commencement of pairing.
Age of the animals at the start of the study (Day 0 of gestation) 77 to 86 days old.
Weight range of the animals at the start of the study (Day 0 of gestation) 176-221 g

Environmental Control
Rodent facility: Limited access - to minimize entry of external biological and chemical agents and to
minimize the transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 20-24ºC and
40-70%.
There were no deviations from these ranges.
Lighting: Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply: Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods. Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.
Cage distribution The cages constituting each group were blocked by group and mounted in batteries.
Bedding Solid bottom cages contained softwood based bark-free fiber bedding (sterilized by autoclaving), which was changed at appropriate intervals each week.
Number of animals per cage Acclimatization up to four animals, During pairing one (stock) male and one female. Gestation one female

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study (except during pairing) and replaced when necessary.
Plastic shelter Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.

Diet Supply
Diet SDS VRF1 Certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability: Non-restricted

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.

Availability: Non-restricted.

Supplier Certificates of Analysis
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was
released for use. Certificates of analysis were routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the softwood based bark-free
fiber bedding and Aspen chew blocks.

No specific contaminants were known that may have interfered with or prejudiced the
outcome of the study and therefore no special assays were performed.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
1 Control 0 mg/kg/day 0 mg/ml dosed at 4ml/kg
2 Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives 10 mg/kg/day, 2.5 mg/ml, dose at 4 ml/kg
3 Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives 30 mg/kg/day, 7.5 mg/ml, dosed at 4 ml/kg
4 Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives 125 mg/kg/day, 31.25 mg/ml, dosed at 4 ml/kg
# Expressed in terms of material as supplied.

Correction factor: None.
Vehicle: Arachis oil.
Method of preparation:
The test item was prepared for administration as a series of graded concentrations in the vehicle. Starting with the low concentration (2.5 mg/mL), the formulation was prepared by weighing out the
required amount of test item and adding approximately 50% of the final volume of vehicle. It was then magnetically stirred. The solution was made up to the required volume with the vehicle and stirred
using a magnetic stirrer until homogenous.

The procedure was repeated for the mid and high dose (7.5 and 31.25 mg/mL).
Frequency of preparation Weekly, and prepared in advance of the first day of dosing.
Storage of formulation: Refrigerated (2 to 8 °C) for up to 20 days.
Test item accounting: Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation Analysis
Stability and homogeneity The homogeneity and stability of formulations during storage was confirmed and supplied by the Sponsor (Harlan Project Nos. 0142-0416 and 0142-0417).

The mean concentrations were within 5% of the nominal concentration, confirming the accuracy of formulation. The difference between the samples remained within 3%, confirming precise analysis.

Achieved concentration: Samples of each formulation prepared for administration Days 6 and 19 after mating were analyzed for achieved concentration of the test item.

Achieved procedural recovery results for both Day 6 and Day 19 were significantly greater than the validated range for this method. A different system was used for these occasions and is consid
ered to be the reason for this, as all results are corrected for the mean procedural recovery value at analysis there is no impact on the results.
Details on mating procedure:
Mating
Male/female ratio 1:1 with identified stock males.

Daily checks for evidence of mating.

Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm. Day 0 of gestation When positive evidence of mating was detected.

A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
Duration of treatment / exposure:
Administration
Route Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.

Treated at Constant doses in mg/kg/day.

Volume dose 4 mL/kg body weight.

Individual dose volume Calculated from the most recently recorded scheduled body weight.

Control (Group 1) Vehicle at the same volume dose as treated groups.

Frequency: Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.

Formulation: A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.

Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
Frequency of treatment:
Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.
Duration of test:
Termination is on Day 20 after mating.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control - Vehicle arachis oil
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 femlaes per dose
1 Control No 1-20
2 10mg/kg/day No 21-40
3 30 mg/kg/day No 41-60
4 125 mg/kg/day No 61-80
Control animals:
yes, concurrent vehicle
Details on study design:
The study consisted of one control and three treated groups.

Animal Model
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Han Wistar (RccHan™:WIST) strain was used because of the historical control data
available at this laboratory.

Route of Administration
The oral (gavage) route of administration was chosen to simulate the conditions of potential human exposure during manufacture, handling or use of the test item.

Rationale for Dose Level Selection
The doses used in this study (0, 10, 30 and 125 mg/kg/day) were selected in conjunction with the Sponsor.

The dose levels were chosen based on the results of a combined repeat dose toxicity study with a reproduction/developmental toxicity screening test repeated OECD 422 study (Harlan Project No.0142 0417). In that study there were no adverse clinical signs and no effects on body weight or food consumption, however slightly increased liver and spleen weights were observed at macroscopic examination. At microscopic examination, acanthosis was seen in the forestomach of males and females treated at 125 mg/kg/day. Since the forestomach present in the rodent is not present in the human stomach and the functionality of the stomach was not compromised, these findings were not considered to be indicative of a risk to human health. Low litter size (due to low corpora lutea and low implantation counts) was evident at 125 mg/kg/day.
Therefore, 125 mg/kg/day was selected as the high dose level in this study. The intermediate and low dose levels of 30 mg/kg/day and 10 mg/kg/day, respectively, were selected to allow evaluation of any dose related trends.

Allocation and Identification

Allocation: On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.

Method: To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups.

Allocation was controlled to prevent any stock male from providing more than one mated female in each treatment group.

Identification of animals : Each animal was assigned a number and identified uniquely within the study by a microchip inserted subcutaneously in the dorsal cervical region.
Identification of cages: Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).
Maternal examinations:
Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were recorded daily at the following times in relation to dose administration:
One to two hours after completion of dosing. As late as possible in the working day.

Clinical Signs
A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

Body Weight
The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

Food Consumption
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

Terminal Investigations
Method of Kill
Method of kill for all adult animals was Carbon dioxide asphyxiation.

Method of kill for fetuses Chilling on a cool plate (approximately 0C).

Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Schedule Animals were killed on Day 20 after mating. Sequence To allow satisfactory inter-group comparison.
Ovaries and uterine content:
The following were recorded for all animals: Uterus Gravid uterine weight (including cervix and ovaries).
For each ovary/uterine horn Number of: Corpora lutea.
Implantation sites.
Resorption sites (classified as early or late).
Fetuses (live and dead).
Apparently non-pregnant animals and for apparently empty uterine horns.

The number of uterine implantation sites were checked after staining with ammonium sulphide [modification of the Salewski staining technique.
Fetal examinations:
Fetal Examination and Processing
Examination of all viable fetuses and placentae, dissected from the uterus, individually weighed and
identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. The sex of each fetus was recorded.

Examination of nominally 50% of fetuses in each litter Sexed internally and eviscerated.

Fixation Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS).

Remaining fetuses were fixed whole in Bouin’s fluid. Processing Bouin’s fixed fetuses were subject to free-hand serial sectioning.

IMS fixed fetuses were processed and stained with Alizarin Red.

Fetal Pathology Examination
Bouin’s fixed fetuses Serial sections were examined for visceral abnormalities.

Alizarin Red stained fetuses Assessed for skeletal development and abnormalities.
Statistics:
Statistical Analysis
The following data types were analyzed at each timepoint separately:
Body weight, using absolute values and gains over appropriate study periods Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
C-section litter data (corpora lutea, implantations, pre/post implantation loss, live
young and sex ratio - percentage male)
Fetal, placental and litter weight
 
No statistical analysis were performed on the results of the fetal examinations only a comparison of incidence with the concurrent and historical controls.
Indices:
Reproductive Assessment
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:

Pre-implantation loss (%) = ((Number of corpora lutea - Number of implantations)/Number of corpora lutea) x 100
Where the number of implantations exceeded the number of corpora lutea observed, pre‑implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).

Post-implantation loss was calculated from the formula:
Post-implantation loss (%) = ((Number of implantations - Number of live fetuses)/Number of implantations) x 100
All group values and SD were calculated from the individual litter values.
Historical control data:
See attached Annex Fetal examination - Major Historical control data
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One female receiving 125 mg/kg/day was observed with rales on Day 20 of gestation. There were no clinical signs that were considered to be associated to treatment with Ethanol,
2,2’-Iminobis-, N-C12-18-Alkyl Derives.

There were no dosing signs recorded, hence no data has been presented.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight performance of females treated with Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives was generally similar to the Controls throughout gestation. See Table 1 and 2 attached.

Body weight gain was slightly lower for females receiving 125 mg/kg/day when compared with Controls.

The mean gravid uterine weight for females treated with Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives was comparable to the mean gravid uterine weight from the Control females.

Adjusted body weight change (0-20) of females treated with 125 mg/kg/day of Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives was statistically significantly low when compared to the Controls
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption for females treated at 125 mg/kg/day of Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives was slightly but statistically significant lower during Days 6-14 of gestation, resulting in a statistically significantly low overall food intake during the treatment period (Days 6-18), when compared with the Control group. See Table 3 attached.

Group mean food intake during Days 10-14 of gestation was slightly but statistically significantly lower for females receiving 30 mg/kg/day when compared with Controls.
Food efficiency:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic findings in dams on Day 20 of gestation that were attributable to treatment
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Post implantation loss (%) was statistically significantly high and total resorptions were higher in females treated at 125 mg/kg/day when compared to the Control group. However, as the number of live fetuses was comparable to the Controls, no definite effect of treatment is inferred.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
On Day 20 of gestation, mean numbers of corpora lutea, implantations, the number of live young and sex ratio were comparable to the Controls, and were considered to be unaffected by treatment with Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives. See Table 4 attached
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
One female (No. 38) treated at 10 mg/kg/day with Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives was found not to be pregnant at macroscopic examination, all other females were pregnant.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Group mean placental weights, litter weights, and male or female fetal weights were all unaffected by treatment with Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives. See Table 5 attached.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
On Day 20 of gestation, the sex ratio was comparable to the Controls, and were considered to be unaffected by treatment with Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Group mean placental weights, litter weights, and male or female fetal weights were all unaffected by treatment with Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives.
Changes in postnatal survival:
not examined
External malformations:
effects observed, treatment-related
Description (incidence and severity):
At 125 mg/kg/day there were 8 litters with similar major abnormalities, the majority affecting the head, eye and vertebral column. These abnormalities include, but are not limited to, Exencephaly/Me
ningoencephalocele/Acephalostomia and Cleft lip; Anophthalmia and Microphthalmia; Spina Bifida/Holorachischisis. Major abnormalities of this combination and severity are rare in rats and the majority are outside of Historical Control Data range.

At 30 mg/kg/day there was an incidence of Microphthalmia (not associated with severe cranial abnormalities). An incidence of 1 fetus in 1 litter is extremely low and is seen at this incidence within the control population as documented within the Historical Control Data range. However, as there were also 2 fetuses in 2 litters with Microphthalmia at 125 mg/kg/day as well as Anophthalmia, a
relationship to treatment cannot be ruled out.

See Tables 6, 7 and 8. and APPENDIX Fetal Examination for individual data.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
At 125 mg/kg/day there were 8 litters with similar major abnormalities, the majority affecting the head, eye and vertebral column. These abnormalities include, but are not limited to, Exencephaly/Meningoencephalocele/Acephalostomia and Cleft lip; Anophthalmia and Microphthalmia; Spina Bifida/Holorachischisis. Major abnormalities of this combination and severity are rare in rats and the majority are outside of Historical Control Data range.

Also, at 125 mg/kg/day there was an increased incidence of medially thickened/kinked ribs, short supernumerary cervical ribs, delayed ossification of 5th/6th sternebrae andthoracic/sacrocaudal vertebral elements and partially undescended lobe(s) of thymus compared to concurrent control.

See Tables 6, 7 and 8. and APPENDIX Fetal Examination for individual data.

The incidences of delayed ossification were within Historical Control Data range. This is an indication of fetal immaturity and as a transient stage in fetal development, not thought to be adverse.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
At 125 mg/kg/day there were 8 litters with similar major abnormalities, the majority affecting the head, eye and vertebral column. These abnormalities include, but are not limited to, Exencephaly/Me
ningoencephalocele/Acephalostomia and Cleft lip; Anophthalmia and Microphthalmia; Spina Bifida/Holorachischisis. Major abnormalities of this combination and severity are rare in rats and the majority are outside of Historical Control Data range.
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
At 125 mg/kg/day there were 8 litters with similar major abnormalities, the majority affecting the head, eye and vertebral column. These abnormalities include, but are not limited to, Exencephaly/Meningoencephalocele/Acephalostomia and Cleft lip; Anophthalmia and Microphthalmia; Spina Bifida/Holorachischisis. Major abnormalities of this combination and severity are rare in rats and the majority are outside of Historical Control Data range.
Dose descriptor:
LOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
visceral malformations
Remarks on result:
other: microphthalmia
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
skeletal malformations
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
external: eye
external: face
skeletal: skull
skeletal: vertebra
visceral/soft tissue: eye
Description (incidence and severity):
At 125 mg/kg/day there were 8 litters with similar major abnormalities, the majority affecting the head, eye and vertebral column. These abnormalities include, but are not limited to, Exencephaly/Meningoencephalocele/Acephalostomia and Cleft lip; Anophthalmia and Microphthalmia; Spina Bifida/Holorachischisis. Major abnormalities of this combination and severity are rare in rats and the
majority are outside of Historical Control Data range.
Also, at 125 mg/kg/day there was an increased incidence of medially thickened/kinked ribs, short
supernumerary cervical ribs, delayed ossification of 5th/6th sternebrae and thoracic/sacrocaudal vertebral elements and partially undescended lobe(s) of thymus compared to concurrent control.
The incidences of delayed ossification were within Historical Control Data range. This is an indication of fetal immaturity and as a transient stage in fetal development, not thought to be adverse.
At 30 mg/kg/day there was an incidence of Microphthalmia (not associated with severe cranial abnormalities). An incidence of 1 fetus in 1 litter is extremely low and is seen at this incidence within the control population as documented within the Historical Control Data range. However, as there were also 2 fetuses in 2 litters with Microphthalmia at 125 mg/kg/day as well as Anophthalmia, a relationship to treatment cannot be ruled out.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
The results from the OECD414 pre-natal development study in rats indicate that Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives produces major skeletal abnormalities in the skull and spinal cord including effects in the eyes of microphthalmia and anophthalmia. The incidence of one fetus in one litter at 30 mg/kg/day is within the historical control range so 30mg/kg/day could be the true NOAEL, however a conservative NOAEL of 10mg/kg/day was selected as it was not possible to be sure that this effect was not treatment related.
Executive summary:

The purpose of this study was to assess the influence of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives, substance used in industry, on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Han Wistar rat.

 

Three groups of 20 females received Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives at doses of 10, 30 or 125 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, arachis oil, at the same volume dose as the treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

 

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

 

Results

The mean concentrations were within 5% of the nominal concentration, confirming the accuracy of formulation. The difference between the samples remained within 3%, confirming precise analysis.

 

Dose levels of 0, 10, 30 and 125 mg/kg/day were well tolerated with no signs observed following dose administration and no adverse clinical signs. At 125 mg/kg/day, minor maternal toxicity was manifest as a slight reduction in mean body weight gain during Days 6-19 and 19-20 of gestation and a statistically significant reduction in adjusted body weight gain (69% of Controls for Day 6-20), and statistically significantly lower group mean food consumption during Days 6-14 of gestation.

 

There was considered to be no adverse effect of maternal treatment upon numbers of implantations, early, late and total resorptions, number of live young and sex ratio and the extent of pre and post-implantation loss. Placental, litter and fetal weights were also all considered to be unaffected by treatment.

 

Fetal development was severely compromised at 125 mg/kg/day, there were 8 litters with similar major abnormalities, the majority affecting the head, eye and vertebral column. At 30 mg/kg/day there was an incidence of Microphthalmia (not associated with severe cranial abnormalities). An incidence of 1 fetus in 1 litter is extremely low and is seen at this incidence within the control population as documented within the Historical Control Data range, which was also observed for 2 fetuses in 2 litters at 125 mg/kg/day, therefore a relationship to treatment at 30 mg/kg/day is uncertain but cannot be ruled out. There were no major abnormalities considered to be related to treatment at 10 mg/kg/day.

 

Conclusion

It was concluded from this study that the dosage of 125 mg/kg/day was the maternal no-observed-adverse-effect-level (NOAEL). Due to the extent and nature of the major abnormalities seen at 125mg/kg/day and the uncertainty of the fetal pathology findings at 30 mg/kg/day, the no-observed-adverse-effect-level (NOAEL) for embryo-fetal survival and development is 10 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Good quality but only a screening study. Annex IX testing will establish if there is an adverse effect requiring classification.
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

Bis(2-hydroxyethyl) coco alkylamine CAS No 61791-31-9 has been tested in the OECD422 test which is a screening study for reproductive toxicity. In this study there were some indications in the top dose of 125 mg/kg/day of developmental toxicity, in particular post-implantation loss indicating possible foetotoxicity. 

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 they still showed some increased post implantation loss individually, the highest loss was of 4 out of 14 implantations, which compared to one negative control female that showed 4 losses out of 17 implantations. 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. 

In the OECD414, dose levels of 0, 10, 30 and 125 mg/kg/day were well tolerated with no signs observed following dose administration and no adverse clinical signs. At 125 mg/kg/day, minor maternal toxicity was manifest as a slight reduction in mean body weight gain during Days 6-19 and 19-20 of gestation and a statistically significant reduction in adjusted body weight gain (69% of Controls for Day 6-20), and statistically significantly lower group mean food consumption during Days 6-14 of gestation.

 

There was considered to be no adverse effect of maternal treatment upon numbers of implantations, early, late and total resorptions, number of live young and sex ratio and the extent of pre- and post-implantation loss. Placental, litter and fetal weights and fetal abnormalities were also all considered to be unaffected by treatment.

 

Fetal development was severely compromised at 125 mg/kg/day, there were 8 litters with similar major abnormalities, the majority affecting the head, eye and vertebral column. At 30 mg/kg/day there was an incidence of Microphthalmia (not associated with severe cranial abnormalities). An incidence of 1 fetus in 1 litter is extremely low and is seen at this incidence within the control population as documented within the Historical Control Data range. However, as there were also 2 fetuses in 2 litters with Microphthalmia at 125 mg/kg/day as well as Anophthalmia, a relationship to treatment cannot be ruled out. There were no major abnormalities considered to be related to treatment at 10 mg/kg/day.

 

It was concluded from this study that the dosage of 125 mg/kg/day was the maternal no-observed-adverse-effect-level (NOAEL). Due to the extent and nature of the major abnormalities seen at 125mg/kg/day and the uncertainty of the fetal pathology findings at 30 mg/kg/day, the no-observed-adverse-effect-level (NOAEL) for embryo-fetal survival and development is 10 mg/kg/day

 

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

This is a full GLP Klimisch 1 OECD422 reproduction screening study carried out with the substance to be registered 2, 2’-(C12-18 evennumbered alkyl imino) diethanol, CAS No 71786-60-2, described at the time of testing as Ethanol, 2, 2’-iminobis-, N-coco alkyl derives CAS No 61791-31-9, these are the same substance.  There were indications of increased post implanation loss at the top dose, in the presence of some maternal toxicity.  As the study is a screening study this was not sufficient for classification.

In addition, there is a full GLP Klimisch 1 OECD414 pre-natal development in rats in this study It was concluded from this study that the dosage of 125 mg/kg/day was the maternal no-observed-adverse-effect-level (NOAEL). Due to the extent and nature of the major abnormalities seen at 125mg/kg/day and the uncertainty of the fetal pathology findings at 30 mg/kg/day, the no-observed-adverse-effect-level (NOAEL) for embryo-fetal survival and development is 10 mg/kg/day. This is the key result when classification for developmental effects is considered.

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.  

Mode of Action Analysis / Human Relevance Framework

There is no specific information on the mode of action leading to the skeletal abnormalities seen in the OECD422 study. There was no evidence of any significant systemic toxicity in the OECD422 study, local irritant effects in the stomach are the limiting factor when setting the oral dose level due to the corrosive properties of the test substance. Molecules like Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derives which have surfactant properties can disrupt cell membranes if they become systemically available. Also there are indication that mono and Diethanolamine are metabolites and these have been shown to reduce choline levels in rats. Choline is a key constituent for the synthesis of cell membranes so this may also be involved. An OECD414 pre-natal development study in rabbits is in progress this will establish if the developmental toxicity is also seen in rabbits. Cross species effects in both rats and rabbits would indicate that the effects must be considered relevant to humans

Justification for classification or non-classification

There are no indications in the OECD422 study at the top dose of 125mg/kg/day of any effects on mating performance gestation period or fertility and no indications to suggest toxic effects on or via lactation. There are also no effects on the reproductive organs seen during the histopathological examinations of the parental rats. Based on the lack of adverse effects there is no reason to propose any classification for effects on fertility or on or via lactation.

 

There were indications in the top dose of 125 mg/kg/day of possible developmental toxicity, specifically increased post-implantation loss indicating possible foetotoxicity. There were also some indications of increased offspring mortality after birth and reduced bodyweight gain. The interpretation of this was complicated by the possible maternal toxicity, of significantly reduced water and food consumption and bodyweight gain during lactation, seen in two of the most affected litters. These females also showed some of the highest post-implantation losses. The OECD422 study is a screening test for reproductive toxicity, due to the study design there are some limitations in interpretation of the results, in addition there were no indications in this study of a dose response for these effects.  Effects on reproduction will be fully investigated in the planned OECD443 Extended one generation reproduction study.

 

In the OECD414 pre-natal development study in rats fetal development was severely compromised at 125 mg/kg/day, there were 8 litters with similar major abnormalities, the majority affecting the head, eye and vertebral column. At 30 mg/kg/day there was an incidence of Microphthalmia (not associated with severe cranial abnormalities). An incidence of 1 fetus in 1 litter is extremely low and is seen at this incidence within the control population as documented within the Historical Control Data range. However, as there were also 2 fetuses in 2 litters with Microphthalmia at 125 mg/kg/day as well as Anophthalmia, a relationship to treatment cannot be ruled out. There were no major abnormalities considered to be related to treatment at 10 mg/kg/day which was selected as the NOAEL.

 

It is concluded that there is very likely a link between the high post implantation loss in the OECD422 and developmental toxicity seen in rats in the OECD414 study. It was decided to continue to base the DNELs on the NOAEL of 30mg/kg from the OECD422 study as this would result in lower more protective DNELs than those based on the 10mg/kg NOAEL from the OECD414 rat study.

 

The adverse effects on development seen in the OECD414 study were sufficient to require classification for developmental toxicity. A rabbit OECD414 pre-natal development study in rabbits is also planned, a final decision on classification will be made when this is completed in 2019. In the interim Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derives has been self-classified as Category 2 for developmental toxicity.