Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: OECD 422 Screening study
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Between 16 October 2009 and 08 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification : Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9)
Description : pale brown viscous liquid
Batch number : S-001016
Date received : 08 July 2009
Storage conditions : approximately 4ºC in the dark, under nitrogen
Expiry date :26 June 2017

Test animals

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)

Administration / exposure

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.






Doses / concentrations
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

Examinations

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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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.

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

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.


Effect levels (F1)

Dose descriptor:
NOEL
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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.

 

 

 

Applicant's summary and conclusion

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

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

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

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

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

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

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

  

Introduction.

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

 

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

 

Methods.

The test material was administered by gavage to three groups each of ten male and ten female Wistar Han™:HsdRccHan™:WIST strain rats, for up to forty-five consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 10, 30 and 125 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males, were treated with the high dose (125 mg/kg/day) or the vehicle alone for forty-two days and then maintained without treatment for a further fourteen days.

 

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

 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

 

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

 

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

 

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

 

Conclusion.

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

 

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