Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
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:
Rational for reliability: 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. Rational for read-across: The registration substance and the read-across supporting substance belong to the category "Primary Fatty Amine Ethoxylates"

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
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 generation)

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
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:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

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)


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

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:
NOAEL
Generation:
F1
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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Tab 1a: Body weight development for males [g]

Dose group

Day numbers relative to start date

 

1

8

15

22

29

36

43

50

57

Control

Mean

318.3

329.7

341.2

353.7

363.5

375.7

385.1

403.8

411.0

S.D.

12.8

16.7

20.3

19.8

21.6

26.3

28.2

21.1

22.3

N

15

15

15

15

15

15

15

5

5

10 mg/kg bw/day

Mean

320.9

336.9

348.0

360.7

371.6

379.7

389.6

-

-

S.D.

10.2

13.3

17.9

20.3

21.3

23.2

24.4

-

-

N

10

10

10

10

9

10

10

0

0

30 mg/kg bw

Mean

325.5

340.1

353.9

365.0

376.5

387.3

398.7

-

-

S.D.

9.8

14.7

18.2

19.7

21.1

21.5

22.4

-

-

N

10

10

10

10

10

10

10

0

0

125 mg/kg bw/day

Mean

320.9

330.4

340.7

353.3

361.5

372.3

382.9

395.2

404.8

S.D.

11.7

15.4

17.8

21.0

20.9

22.8

23.8

20.3

23.0

N

15

15

15

15

15

15

15

5

5

Tab 1b: Body weight development for females [g]

Dose group

 

Pre-mating

Gestation

Lactation

 

1

8

15

0

7

14

20

1

4

Control

Mean

207.8

211.6

217.9

218.0

243.2

268.6

333.8

235.1

248.6

S.D.

7.3

3.6

7.3

7.9

10.1

14.7

18.9

11.9

11.4

N

10

10

10

10

10

10

10

10

10

10 mg/kg bw/day

Mean

205.6

207.4

213.7

214.9

240.4

267.7

327.0

247.0

257.1

S.D.

12.7

9.7

14.4

11.2

15.7

19.2

24.8

23.0

21.1

N

10

10

10

9

9

9

9

9

9

30 mg/kg bw

Mean

210.6

212.8

217.7

224.1

246.0

272.9

339.8

243.8

256.6

S.D.

10.2

8.9

12.6

12.7

11.9

10.9

16.7

14.6

13.5

N

10

10

10

10

10

10

10

10

10

125 mg/kg bw/day

Mean

212.3

211.4

218.6

222.0

243.6

269.3

321.9

259.3

260.1

S.D.

9.6

11.7

11.3

14.2

15.2

17.7

24.2

18.1

18.5

N

10

10

10

9

9

9

9

9

9

Tab 2: Hematology for males (N=5) at termination and after recovery:

- data for females not shown as no difference was found for control and treated animals

Dose group

 

Hb

RBC

Hct

MCH

MCV

MCHC

WBC

Neut

Lymph

CT

PLT

APTT

Retics

 

g/dl

10^12/l

%

pg

g/dl

g/dl

10^9/l

10^9/l

10^9/l

sec

10^9/l

sec

%

At termination

Control

Mean

16.60

8.974

48.68

18.48

54.28

34.06

7.56

0.902

6.562

11.06

530.4

16.84

4.80

S.D.

0.41

0.386

1.17

0.42

1.34

0.38

1.29

0.280

1.026

0.89

77.3

0.81

0.71

10 mg/kg bw/day

Mean

16.28

9.158

46.60

17.80

50.88**

34.96

6.66

0.744

5.858

11.72

581.4

16.68

4.56

S.D.

0.27

0.367

1.15

0.82

1.26

1.23

1.61

0.190

1.635

1.41

73.2

1.32

0.47

30 mg/kg bw/day

Mean

16.62

9.176

48.32

18.10

52.60**

34.44

7.98

0.966

6.942

12.36

577.8

16.84

4.68

S.D.

0.77

0.362

2.71

0.19

1.13

0.58

1.73

0.523

1.528

1.99

33.5

1.27

0.40

125 mg/kg bw/day

Mean

15.60**

8.854

45.62*

17.62*

51.52**

34.20

8.06

0.756

7.206

12.86

632.2*

16.62

5.78*

S.D.

0.34

0.240

0.80

0.11

1.26

0.64

1.70

0.336

1.833

2.29

58.4

1.11

1.02

After recovery

Control

Mean

15.66

8.962

47.64

17.48

53.20

32.88

7.22

0.778

6.356

9.12

638.0

13.30

4.58

S.D.

0.39

0.312

1.43

0.51

1.67

0.18

0.75

0.160

0.717

0.41

46.9

1.22

0.40

50 mg/kg bw/day

Mean

15.40

9.168

46.76

16.80*

51.00*

32.94

7.64

1.042

6.512

9.12

690.8

14.66

5.54

S.D.

0.66

0.376

2.35

0.14

0.80

0.34

0.36

0.283

0.432

0.16

90.8

0.72

0.91

Tab 3: Organ weight for males and females [g]:

-data shown only for organs with statistical differences for control and treated animals

 

males

females

 

At treatment termination

After recovery of 14 days

At treatment termination

Dose group[g]

Dose group[g]

Dose group[g]

Control

10

30

125

0

125

Control

10

30

125

Terminal body weight

Mean

380.8

389.6

398.7

382.5

411.0

404.8

250.0

257.0

255.4

259.2

S.D.

31.5

24.4

22.4

25.2

22.3

23.0

8.1

24.5

12.8

18.5

N

10

10

10

10

5

5

10

9

10

9

Kidneys

Mean

1.99

2.18*

2.17*

2.15*

2.24

2.24

1.43

1.42

1.52

1.53

S.D.

0.20

0.19

0.13

0.16

0.13

0.18

0.10

0.08

0.08

0.11

N

10

10

10

10

5

5

10

9

10

9

Liver

Mean

11.97

12.34

12.38

13.40**

12.45

12.34

10.59

10.19

10.40

11.43

S.D.

1.20

0.87

0.90

1.13

1.79

0.56

1.26

1.02

0.86

1.13

N

10

10

10

10

5

5

10

9

10

9

Spleen

Mean

0.64

0.63

0.68

0.73*

0.66

0.71*

0.62

0.63

0.63

0.63

S.D.

0.06

0.09

0.09

0.11

0.07

0.03

0.06

0.09

0.09

0.09

N

10

10

10

10

5

5

10

9

10

9

Thyroid

Mean

0.012

0.014

0.017*

0.016*

0.023

0.020

0.014

0.015

0.015

0.017

S.D.

0.004

0.003

0.005

0.004

0.014

0.005

0.003

0.006

0.004

0.007

N

10

10

10

10

5

5

10

9

10

9

Tab 4: Summary report of effects on reproduction/development

 

Dose group

 

Control

10

mg/kg bw/day

30

mg/kg bw/day

125

mg/kg bw/day

No. of females paired

10

10

10

10

No. of females mated

10

10

10

10

No. of females pregnant

10

9

10

10

No. of dams with living pubs

10

9

10

9

Body weight on GD20 [g]

334

327

340

322

Body weight on PN4 [g]

249

257

257

260

Corpora lutea

19.4

16.7

18.2

15.2

Implantation sites

15.6

12.4

14.3

  12.3*

No. of pubs alive at birth

14.3

11.9

13.6

       8.8***

No. of pubs alive at PN4

14.0

11.8

12.5

       7.7***

Applicant's summary and conclusion

Conclusions:
The reproduction toxicity of the registration substance is derived based on the read-across approach.
The supporting substance CAS 71786 -60 -2 was tested for its reproduction toxicity according to OECD 422 Test Guideline and the NOAEL of 30 mg/kg/day was obtained. The major effecst were changes in forestomach and in stomach at 30 and 125 mg/kg/day and increased implantation loss and decreased number of live pubs at 125 mg/kg bw.
Executive summary:

The reproduction toxicity of the registration substance is derived based on the read-across approach.

Bis (2-hydroxyethyl) coco alkylamineCAS 71786 -60 -2, was tested for its reproduction toxicity according to OECD 422 Test Guideline.The rats were treated at doses of 0, 10, 30 and 125 mg/kg/day for up to forty five days. The irritant effect such as salivation and histopathological alteration in the gastric tract was evident at 125 mg/kg/day. Further, a possible effect on the hematopoietic system could be derived based on the minimal changes in hematology parameters and the slightly increased spleen weights in males. Also the liver weight was minimally increased for males that was considered as adaptive responce. Based on the histopathological changes observed in the stomach at 125 mg/kg/day the NOAEL of 30 mg/kg/day was derived for the systemic toxicity.

At dose of 125 mg/kg bw lower litter size was evident. Due to the findings in the parental animals the reproduction effects at 125 mg/mg/day is likely to be secondary to the maternal effect. The NOAEL of 30 mg/kg/day was derived for the reproductive toxicity.