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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
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: 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.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test”
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

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:
Wistar
Details on test animals and environmental conditions:
Test Animals
- Source:
Wistar Han™:HsdRccHan™:WIST strain rats from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.

- Age at study initiation:
Approximately 12 weeks old

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

- Housing:
Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

- Diet:
The animals were allowed free access to food. A pelleted diet Rodent 2018C
Teklad Global Certified Diet Harlan Laboratories U.K. Ltd., Oxon, UK was used throughout the study period. The diet was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

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

- Acclimation period:
For 12 days

ENVIRONMENTAL CONDITIONS

- Temperature:
21 ± 2 °C

- Humidity:
55 ± 15 %

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

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

IN-LIFE DATES:
Up to 45 consecutive days for females and 42 days for males.



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 +/- 9% of the nominal concentration.

DIET PREPARATION
- Not applicable

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

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

- Storage temperature of food:
No data

VEHICLE
Arachis oil BP

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

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

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

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

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

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

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

The analytical method has been satisfactorily validated in terms of specificity and accuracy for the purposes of the study.

See attached Appendix 26 - Chemical Analysis of Test Material Formulations, Methods
Details on mating procedure:

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

- Length of cohabitation:
Up to 14 days

- Proof of pregnancy:
Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation)

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

- Further matings after two unsuccessful attempts:
Not applicable

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

- Any other deviations from standard protocol:
Not applicable
Duration of treatment / exposure:
Non-recovery males from all treatment groups were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

(Following fourteen days without treatment, recovery control and high dose group males were terminated).

Frequency of treatment:
Daily
Duration of test:
The in-life phase of the study was conducted between 20 October 2009 (first day of treatment) and 15 December 2009 (final necropsy).
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-Finding Toxicity Study in the Rat (0142-0416)

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

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

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

- Section schedule rationale (if not random):
Random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:

- Yes see attached tables and appendices

- Time schedule:

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

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

NEUROBEHAVIOURAL EXAMINATION:

- Yes see attached tables and appendices

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

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

BODY WEIGHT:

- Yes see attached tables and appendices

- Time schedule for examinations:

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

- For parameters checked see attached Tables.

FOOD CONSUMPTION:

- Yes see attached tables and appendices

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

- FOOD EFFICIENCY:

- Yes see attached tables

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

WATER CONSUMPTION:

- Yes see attached tables

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

HAEMATOLOGY AND CLINICAL CHEMISTRY:

- Yes see attached tables and appendices

- Time schedule for collection of blood:

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

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

- Anaesthetic used for blood collection:
- No

- Animals fasted:
- No

URINALYSIS:
No

- Time schedule for collection of urine:
Not applicable

- Metabolism cages used for collection of urine:
Not applicable

- Animals fasted:
Not applicable

- Parameters examined:
Not applicable

OTHER:

MATING

- Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

PREGNANCY AND PARTURITION

- Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:

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

LITTER SIZE

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

For each litter the following was recorded:

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

PHYSICAL DEVELOPMENT

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

- see attached tables and appendices

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

- Number of corpora lutea:
Yes

- Number of implantations:
Yes

- Number of early resorptions:
No

- Number of late resorptions:
No

- Other:
Parameters in Table 1 were examined in this study.
Fetal examinations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum:
No

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

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

POST-MORTEM EXAMINATION
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Necropsy findings checked in table 21 were included. All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:
The volume of statistical references exceeds the storage capacity in this section it has therefore been included as an attachment titled 0142-0417 Statistics.
Indices:
Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating
period of the parental generation.
i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive
evidence of mating.
ii) Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals paired ÷ Number of animals mated) x 100
Pregnancy Index (%) = (Number of animals mated ÷ Number of pregnant females) x 100
Gestation and Parturition Data
The following parameters were calculated for individual data during the gestation and
parturition period of the parental generation.
i) Gestation Length
Calculated as the number of days of gestation including the day for observation of
mating and the start of parturition.
ii) Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of pregnant females ÷ Number of females delivering live offspring) x 100
Historical control data:
Included: see Addenum 3-7

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

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

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

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

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

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

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

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

Food Consumption
Group mean food consumptions are given in Table 9 and are presented graphically in Figure 3 and Figure 4. Weekly food efficiencies for males, and for females during the pre-mating phase, are given in Table 10. Individual and group mean food consumptions for females following mating and during lactation are presented in Appendix 7.
No adverse effects on dietary intake or food conversion efficiency (the ratio of bodyweight gain/dietary intake) were evident for males during the treatment or recovery phases of the study.
No adverse effects on dietary intake were evident for females during the pre-mating and gestation phases of the study. A slight reduction in dietary intake was evident for females treated with 125 mg/kg/day when compared to controls during lactation. Statistical analysis of the data for females during gestation and lactation however did not reveal any significant intergroup differences.
Food conversion efficiency during the pre-mating phase was not affected.

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

Reproductive Performance

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

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

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

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

Offspring Litter Size and Viability
Group mean corpora lutea and implantation counts, litter size, implantation losses, survival indices and sex ratio are given in Tables 13 to 15 (statistically significant differences are indicated). Individual data are given in Appendices 10 to 12.
Lower numbers of corpora lutea and implantation sites were evident for females treated with 125 mg/kg/day when compared to controls, although statistical significance was achieved for lower numbers of implantation sites only, when compared to controls (P<0.05). A higher percentage of post-implantation losses was also evident at 125 mg/kg/day when compared to controls (P<0.01). This resulted in significantly lower litter sizes observed at birth for females treated with 125 mg/kg/day when compared to controls (P<0.001). Litter sizes on Day 1 and Day 4 of lactation were therefore also significantly smaller at 125 mg/kg/day when compared to controls (P<0.001). Lower live birth and viability indices were also evident at 125 mg/kg/day when compared to controls, although statistical analysis of the data did not reveal any significant intergroup differences.
Two litters from the 125 mg/kg/day dose group and three litters from the 30 mg/kg/day dose group showed dead offspring at birth. There were no offspring found dead at birth from the control or 10 mg/kg/day litters. A higher incidence of missing offspring (cannabalised by the mother following death) was evident at 125 mg/kg/day and possibly at 30 mg/kg/day in comparison to controls, although statistical analysis of this data did not reveal any significant intergroup differences.
The percentage of male offspring in the 125 mg/kg/day dose group was slightly lower than the number of male offspring observed in the control group, although statistical analysis was not achieved.
No treatment-related effects were evident for litters from the 10 mg/kg/day dose group.
A significantly lower number of implantation sites were noted at 10 mg/kg/day compared to controls (P<0.01). In isolation and in the absence of a dose-related response, this finding was not considered to be of any toxicological importance.

Offspring Growth and Development
Group values for offspring bodyweight and bodyweight change, surface righting reflex and the incidence of clinical signs are given in Tables 13, 16 and 17 (statistically significant differences are indicated). Individual values and observations are given in Appendices 10, 13 and 14.
Total litter weights were lower at 125 mg/kg/day on Day 1 (P<0.01) and Day 4 of lactation (P<0.001) in comparison to control values. Bodyweights for offspring from treated animals were essentially similar to controls and no significant differences in bodyweight gains were evident between Day 1 and Day 4 of lactation.
No treatment-related clinical signs were detected. The clinical signs observed were low incidence findings commonly observed in reproductive studies of this type and unrelated to test material toxicity. Surface righting was not affected at any treatment level.

Laboratory Investigations

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

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

Pathology

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

Necropsy
A summary incidence of necropsy findings is given in Table 21 and Table 22. Individual data are given in Appendices 22 and 23.
Offspring
No treatment-related macroscopic abnormalities were detected for offspring dying during lactation or at termination on Day 5 post partum.
No treatment-related macroscopic abnormalities were detected at terminal kill. The macroscopic abnormalities observed for interim death offspring consisted of autolytic changes, cannibalism, no milk present in the stomach and light brown colouration of the liver. Remaining macroscopic findings observed at termination were considered to be low incidence findings occasionally observed in reproductive studies of this type, and not related to test material toxicity.
Adults
Treatment-related findings were confined to the presence of a thickened glandular and non-glandular region of the stomach recorded for one non-recovery male treated with 125 mg/kg/day at the end of the treatment period.
There were no further macroscopic abnormalities which were considered to be attributable to treatment with the test material.
A reddened median lobe of the liver was evident for one male treated with 30 mg/kg/day. Sloughing of the glandular gastric epithelia was observed for one female treated with 10 mg/kg/day, although sloughing of the glandular and non-glandular epithelia was also recorded for one control female. In the absence of any histopathological changes in the stomach observed at this dose level, the toxicological importance of this finding was minimal.

Histopathology
A summary incidence of histopathological findings is given in Table 23. Individual data are given in Appendix 24.
The following treatment-related changes were observed:
STOMACH: Acanthosis, frequently with associated hyperkeratosis, was seen in the forestomach of all animals of either sex treated with 125 mg/kg/day, and in males only treated with 30 mg/kg/day. There was evidence of regression of the condition in recovery 125 mg/kg/day males following an additional fourteen days without treatment, with just two animals affected.
Remaining histopathological changes seen among surviving control and high dose animals were all considered to be spontaneous in origin and unrelated to treatment. The following conditions warrant specific mention:
ADRENAL GLANDS: Cortical vacuolation was seen in a few control and treated males and was of no toxicological significance in this investigation.
BONE MARROW: Adipose infiltration of the marrow is an indicator of changes in marrow cellularity and in this study, there was no difference between control and treated groups.
KIDNEYS: Focal corticomedullary mineralisation is a commonly observed background condition among females. Globular accumulations of eosinophilic material, as a consequence of excessive accumulation of alpha2-microglobulin in renal proximal tubular epithelial cells, are occasionally encountered as a spontaneous change in male rats.
LIVER: Scattered mononuclear cell foci were observed in a few control and treated animals examined in the study. Such are commonly observed in the rodent liver and are not indicative of any adverse condition at the severities encountered.
LUNGS: A minimal severity of bronchus associated lymphoid tissue was reported for all control and high dose animals examined in the study and is not indicative of respiratory disease. Minor severities and low incidences of focal pneumonitis and accumulations of alveolar macrophages are commonly observed pulmonary changes in laboratory maintained rats of this age and are not suggestive of significant respiratory disease.
SPLEEN: Extramedullary haemopoiesis is a normal background condition in the rat spleen and the severities observed were considered to be within normal limits.
THYROID: Follicular cell hypertrophy is commonly seen among untreated animals of either sex and there was no indication of a relationship to treatment in this study.
THYMUS: Lymphoid atrophy is frequently seen among pregnant and lactating female rats.
REPRODUCTIVE TRACT AND RELATED ORGANS
PITUITARY: No treatment-related changes were seen. Vacuolation of pars anterior cells is a commonly observed change more especially in male rats.
TESTIS/EPIDIDYMIS: Testicular atrophy is observed occasionally as a spontaneous condition frequently with associated cellular debris in the epididymis.
SEMINAL VESICLES/COAGULATING GLAND: No treatment-related changes were seen.
PROSTATE: No pathological changes were seen.
MAMMARY GLAND: Glandular hyperplasia was observed in the mammary tissue of all previously pregnant females and is consistent with pregnancy and lactation.
OVARY: No pathological changes were seen.
UTERUS/VAGINA: Areas of haemorrhage and fibrosis were seen in the myometrium and adjacent connective tissue of the uterus in the majority of females examined from control and high dose groups. These conditions are consistent with normal post partum uterine changes in the rat. Keratinisation of the uterine cervix and/or vagina is a normal cyclical change in female rats.
All other morphological changes in the above and remaining tissues were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: other:
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

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

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

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

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

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

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

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

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

SEXUAL MATURATION (OFFSPRING)
Not applicable

ORGAN WEIGHTS (OFFSPRING)
Not applicable

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

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

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

HISTOPATHOLOGY (OFFSPRING)
Not applicable

OTHER FINDINGS (OFFSPRING)
Offspring Litter Size and Viability

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

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

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

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

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental 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) (Harlan Laboratories Ltd., Project Number 0142 -0416)

Part 1: Maximum Tolerated Dose Results:

 

Mortality.

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

 

Clinical Observations.

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

 

Bodyweight.

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

 

Necropsy.

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

 

Conclusion.

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

 

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

 

RESULTS

 

Mortality

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

 

Clinical Observations

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

 

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

 

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

75 mg/kg/day.

 

Bodyweight

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

 

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

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

 

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

 

Food Consumption

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

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

 

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

 

Water Consumption

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

 

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

 

Organ Weights

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

 

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

 

Necropsy

 

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

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

 

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

 

DISCUSSION

 

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

 

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

 

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

 

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

 

CONCLUSION

 

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

 

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

 

 

 

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 4 post partum.

 

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

 

Conclusion.

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

 

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