2,2'-(C12-14 evennumbered alkyl imino) diethanol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

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 presence of adverse effects in parental animals, the reproduction effects at 125 mg/kg bw is likely to be secondary to the maternal effect. The NOAEL of 30 mg/kg/day was derived for the reproductive toxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 16 October 2009 and 08 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Wistar Han™:HsdRccHan™:WIST strain rat

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test Animals
- Source:
Wistar Han™:HsdRccHan™:WIST strain rats from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.

- Age at study initiation:
Approximately 12 weeks old

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

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

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

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

- Acclimation period:
For 12 days

ENVIRONMENTAL CONDITIONS

- Temperature:
21 ± 2 °C

- Humidity:
55 ± 15 %

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

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

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

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: Not applicable

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test material formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services (Harlan Laboratories Ltd. Project Number: 0142-0416). Results from the previous study showed the formulations to be stable for at least twenty days. Formulations were therefore prepared twice monthly during the treatment period and stored at approximately +4ºC in the dark, under nitrogen.
Samples of each test material formulation were taken and analysed for concentration of test material at Harlan Laboratories Ltd., Shardlow, UK Analytical Services. The method used for analysis of formulations and the results obtained are given in Appendix 26. The results indicate that the prepared formulations were within plus or minus 9% of the nominal concentration.

DIET PREPARATION
- Not applicable

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

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

- Storage temperature of food:
No data

VEHICLE
Arachis oil BP

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

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

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

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

- Purity:
Not applicable

Details on mating procedure:

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

- Length of cohabitation:
Up to 14 days

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

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

- Further matings after two unsuccessful attempts:
Not applicable

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

- Any other deviations from standard protocol:
Not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9) in the test material formulations was determined by gas chromatography (GC) using an external standard technique.

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

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

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

Duration of treatment / exposure:

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

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

Frequency of treatment:

Daily

Details on study schedule:

Non-Recovery Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.

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

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

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

Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.

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

Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.

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

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

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

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

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

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

Remarks:

Doses / Concentrations:
Dose levels of 10, 30 and 125 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

0 mg/kg/day – control: 10 animals per sex.
10 mg/kg/day : 10 animals per sex.
30 mg/kg/day : 10 animals per sex.
125 mg/kg/day : 10 animals per sex.
Recovery (Satellite ) 0 mg/kg/day – control: 5 males only.
Recovery (Satellite ) 125 mg/kg/day : 5 males only.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on Preliminary Fourteen Day Repeated Dose Oral (Gavage) Range-Finder in the Rat

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

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

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

- Section schedule rationale (if not random):
Random

Positive control:

Not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:

- Yes see attached tables and appendices

- Time schedule:

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

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

NEUROBEHAVIOURAL EXAMINATION:

- Yes see attached tables and appendices

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

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

BODY WEIGHT:

- Yes see attached tables and appendices

- Time schedule for examinations:

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

- For parameters checked see attached Tables.

FOOD CONSUMPTION:

- Yes see attached tables and appendices

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

- FOOD EFFICIENCY:

- Yes see attached tables

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

WATER CONSUMPTION:

- Yes see attached tables

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

HAEMATOLOGY AND CLINICAL CHEMISTRY:

- Yes see attached tables and appendices

- Time schedule for collection of blood:

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

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

- Anaesthetic used for blood collection:
- No

- Animals fasted:
- No

URINALYSIS:
No

- Time schedule for collection of urine:
Not applicable

- Metabolism cages used for collection of urine:
Not applicable

- Animals fasted:
Not applicable

- Parameters examined:
Not applicable

OTHER:

MATING

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

PREGNANCY AND PARTURITION

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

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

LITTER SIZE

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

For each litter the following was recorded:

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

PHYSICAL DEVELOPMENT

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

- see attached tables and appendices

Oestrous cyclicity (parental animals):

A vaginal smear was prepared for each female and the stage of the oestrous cycle was recorded.

Sperm parameters (parental animals):

Parameters examined in all male parental generations:testis During histopathology, the male epididymides were examined for spermatocoel granuloma formation.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum:
No

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

GROSS EXAMINATION OF DEAD PUPS:
Dying offspring during the study were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Postmortem examinations (parental animals):

SACRIFICE

- Male animals:
Adult surviving males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43.

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

GROSS NECROPSY / ORGAN WEIGHTS

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

HISTOPATHOLOGY

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

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

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

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

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

All tissues were despatched to Harlan Laboratories Ltd, Switzerland (Principal Investigator: K Weber). The tissues from five selected control, 150 and 500 mg/kg/day dose group animals and those animals dying during the study, were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control, 150 and 500 mg/kg/day were also processed. Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of kidney and spleen from five animals per sex from the low dose groups.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.

Postmortem examinations (offspring):

Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Necropsy findings checked in table 28 were included. All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

The volume of statistical references exceeds the storage capacity in this section it has therefore been included as an attachment titled 0142-0417 Statistics.

Reproductive indices:

Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating
period of the parental generation.
i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive
evidence of mating.
ii) Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals paired ÷ Number of animals mated) x 100
Pregnancy Index (%) = (Number of animals mated ÷ Number of pregnant females) x 100
Gestation and Parturition Data
The following parameters were calculated for individual data during the gestation and
parturition period of the parental generation.
i) Gestation Length
Calculated as the number of days of gestation including the day for observation of
mating and the start of parturition.
ii) Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of pregnant females ÷ Number of females delivering live offspring) x 100

Offspring viability indices:

The standard unit of assessment was considered to be the litter, therefore values were
first calculated for each litter and the group mean was calculated using their individual
litter values. Group mean values included all litters reared to termination (Day 5 of age).
i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for
each female/litter as follows:
% pre – implantation loss = [(Number of corpora lutea - Number of Corpora Lutea) ÷ Number of implantation sites] x 100
% post – implantation loss =[(Number of implantation sites - Number of implantation sites) ÷ Total number of offspring born] x 100
ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring born ÷Number of offspring alive on Day 1) x 100
Viability Index 1 (%) = (Number of offspring alive on Day 1 ÷ Number of offspring alive on Day 4) x 100
iii) Sex Ratio (% males)
Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula:
(Number of male offspring ÷ Total number of offspring) x 100

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422 1996)

RESULTS:

Mortality.

No unscheduled deaths were detected.

Clinical Observations.

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

Functional Observations.

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

Bodyweight.

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

Food Consumption.

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

Water Consumption.

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

Reproductive performance.

Mating.

No treatment-related effects were detected in mating performance.

Fertility.

No treatment-related effects were detected in fertility.

Gestation.

No treatment-related effects were detected on gestation length.

Litter responses.

Litter size and Viability.

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

Offspring Growth and Development.

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

Laboratory Investigations.

Haematology.

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

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

Blood Chemistry.

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

Pathology.

Organ Weights.

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

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

Necropsy.

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

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

Histopathology. The following treatment-related changes were observed:

STOMACH: Acanthosis, frequently with associated hyperkeratosis, was seen in the forestomach of all animals of either sex treated with 125 mg/kg/day, and in males treated with 30 mg/kg/day. There was evidence of regression of the condition in recovery 125 mg/kg/day males following an additional fourteen days without treatment.

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

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

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.

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

Reproductive effects observed:

not specified

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***

Conclusions:

Bis (2-hydroxyethyl) coco alkylamine, 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:

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.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

other information

Justification for type of information:

The registration substance (target) and the source substance are members of Primary Fatty Amine Ethoxylates Category (see the category justification provided in Chapter 13).

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable and robust read-across based on the category formation

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

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

The supporting substance CAS 71786 -60 -2 was tested for its developmental toxicity according to the OECD Guideline 414.

Pregnant rats were treated daily from gestation day 6 to 19 at doses of 125, 30 and 10 mg/kg/day. Severe maternal toxicity was not found up to the highest dose.

At 125 mg/kg/day fetal toxicity was found (skeletal abnormalities in the skull and spinal cord and effects in the eyes). Also at 30 mg/kg/day the eye of one fetuses was affected. The

NOAEL of 125 mg/kg/day for maternatl toxicity and NOAEL of 10 mg/kg/day for developmental toxicity was obtained.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

other information

Justification for type of information:

The registration substance (target) and the source substance are members of Primary Fatty Amine Ethoxylates Category (see the category justification provided in Chapter 13).

Reason / purpose for cross-reference:

read-across source

Conclusions:

NOAEL 125 mg/kg/day for maternal toxicity; NOAEL 10 mg/kg/day for developmental toxicity

Executive summary:

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

The supporting substance CAS 71786 -60 -2 was tested for its developmental toxicity according to the OECD Guideline 414.

Pregnant rats were treated daily from gestation day 6 to 19 at doses of 125, 30 and 10 mg/kg/day. Severe maternal toxicity was not found up to the highest dose.

At 125 mg/kg/day fetal toxicity was found (skeletal abnormalities in the skull and spinal cord and effects in the eyes). Also at 30 mg/kg/day the eye of one fetuses was affected. The

NOAEL of 125 mg/kg/day for maternatl toxicity and NOAEL of 10 mg/kg/day for developmental toxicity was obtained.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27th March 2018 -12th June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Test item: Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives
Test item identity (including alternative names):
Ethomeen C/12
2,2’-(C12-18 even numbered alkyl imino)diethanol.
Bis(2-hydroxyethyl)cocoalkylamine.
CAS number: 71786-60-2 and 61791-31-9
Intended use: Substance used in industry.
Appearance: Light yellow liquid.
Storage conditions: At ambient temperature (15 to 25C), in the dark.
Supplier: Sponsor
Batch number: 1373142
Stability/expiry date: 28 November 2018
Purity: 98%

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Strain/Species RccHan™:WIST rat.
Supplier Envigo (RMS) UK Limited
Number of animals ordered 90 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization Five days before commencement of pairing.
Age of the animals at the start of the study (Day 0 of gestation) 77 to 86 days old.
Weight range of the animals at the start of the study (Day 0 of gestation) 176-221 g

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

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

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

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

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

Availability: Non-restricted.

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

No specific contaminants were known that may have interfered with or prejudiced the
outcome of the study and therefore no special assays were performed.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

1 Control 0 mg/kg/day 0 mg/ml dosed at 4ml/kg
2 Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives 10 mg/kg/day, 2.5 mg/ml, dose at 4 ml/kg
3 Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives 30 mg/kg/day, 7.5 mg/ml, dosed at 4 ml/kg
4 Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives 125 mg/kg/day, 31.25 mg/ml, dosed at 4 ml/kg
# Expressed in terms of material as supplied.

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

The procedure was repeated for the mid and high dose (7.5 and 31.25 mg/mL).
Frequency of preparation Weekly, and prepared in advance of the first day of dosing.
Storage of formulation: Refrigerated (2 to 8 °C) for up to 20 days.
Test item accounting: Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation Analysis
Stability and homogeneity The homogeneity and stability of formulations during storage was confirmed and supplied by the Sponsor (Harlan Project Nos. 0142-0416 and 0142-0417).

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

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

Achieved procedural recovery results for both Day 6 and Day 19 were significantly greater than the validated range for this method. A different system was used for these occasions and is consid
ered to be the reason for this, as all results are corrected for the mean procedural recovery value at analysis there is no impact on the results.

Details on mating procedure:

Mating
Male/female ratio 1:1 with identified stock males.

Daily checks for evidence of mating.

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

A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.

Duration of treatment / exposure:

Administration
Route Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.

Treated at Constant doses in mg/kg/day.

Volume dose 4 mL/kg body weight.

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

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

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

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

Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Frequency of treatment:

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

Duration of test:

Termination is on Day 20 after mating.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control - Vehicle arachis oil

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Dose / conc.:

125 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20 femlaes per dose
1 Control No 1-20
2 10mg/kg/day No 21-40
3 30 mg/kg/day No 41-60
4 125 mg/kg/day No 61-80

Control animals:

yes, concurrent vehicle

Details on study design:

The study consisted of one control and three treated groups.

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

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

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

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

Allocation and Identification

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

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

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

Identification of animals : Each animal was assigned a number and identified uniquely within the study by a microchip inserted subcutaneously in the dorsal cervical region.
Identification of cages: Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Maternal examinations:

Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

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

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

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

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

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

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

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

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

Schedule Animals were killed on Day 20 after mating. Sequence To allow satisfactory inter-group comparison.

Ovaries and uterine content:

The following were recorded for all animals: Uterus Gravid uterine weight (including cervix and ovaries).
For each ovary/uterine horn Number of: Corpora lutea.
Implantation sites.
Resorption sites (classified as early or late).
Fetuses (live and dead).
Apparently non-pregnant animals and for apparently empty uterine horns.

The number of uterine implantation sites were checked after staining with ammonium sulphide [modification of the Salewski staining technique.

Fetal examinations:

Fetal Examination and Processing
Examination of all viable fetuses and placentae, dissected from the uterus, individually weighed and
identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. The sex of each fetus was recorded.

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

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

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

IMS fixed fetuses were processed and stained with Alizarin Red.

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

Alizarin Red stained fetuses Assessed for skeletal development and abnormalities.

Statistics:

Statistical Analysis
The following data types were analyzed at each timepoint separately:
Body weight, using absolute values and gains over appropriate study periods Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
C-section litter data (corpora lutea, implantations, pre/post implantation loss, live
young and sex ratio - percentage male)
Fetal, placental and litter weight
 
No statistical analysis were performed on the results of the fetal examinations only a comparison of incidence with the concurrent and historical controls.

Indices:

Reproductive Assessment
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:

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

Post-implantation loss was calculated from the formula:
Post-implantation loss (%) = ((Number of implantations - Number of live fetuses)/Number of implantations) x 100
All group values and SD were calculated from the individual litter values.

Historical control data:

See attached Annex Fetal examination - Major Historical control data

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One female receiving 125 mg/kg/day was observed with rales on Day 20 of gestation. There were no clinical signs that were considered to be associated to treatment with Ethanol,
2,2’-Iminobis-, N-C12-18-Alkyl Derives.

There were no dosing signs recorded, hence no data has been presented.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight performance of females treated with Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives was generally similar to the Controls throughout gestation. See Table 1 and 2 attached.

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

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

Adjusted body weight change (0-20) of females treated with 125 mg/kg/day of Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives was statistically significantly low when compared to the Controls

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption for females treated at 125 mg/kg/day of Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives was slightly but statistically significant lower during Days 6-14 of gestation, resulting in a statistically significantly low overall food intake during the treatment period (Days 6-18), when compared with the Control group. See Table 3 attached.

Group mean food intake during Days 10-14 of gestation was slightly but statistically significantly lower for females receiving 30 mg/kg/day when compared with Controls.

Food efficiency:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic findings in dams on Day 20 of gestation that were attributable to treatment

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Post implantation loss (%) was statistically significantly high and total resorptions were higher in females treated at 125 mg/kg/day when compared to the Control group. However, as the number of live fetuses was comparable to the Controls, no definite effect of treatment is inferred.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

On Day 20 of gestation, mean numbers of corpora lutea, implantations, the number of live young and sex ratio were comparable to the Controls, and were considered to be unaffected by treatment with Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives. See Table 4 attached

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

One female (No. 38) treated at 10 mg/kg/day with Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives was found not to be pregnant at macroscopic examination, all other females were pregnant.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Group mean placental weights, litter weights, and male or female fetal weights were all unaffected by treatment with Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives. See Table 5 attached.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

On Day 20 of gestation, the sex ratio was comparable to the Controls, and were considered to be unaffected by treatment with Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Group mean placental weights, litter weights, and male or female fetal weights were all unaffected by treatment with Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives.

Changes in postnatal survival:

not examined

External malformations:

effects observed, treatment-related

Description (incidence and severity):

At 125 mg/kg/day there were 8 litters with similar major abnormalities, the majority affecting the head, eye and vertebral column. These abnormalities include, but are not limited to, Exencephaly/Me
ningoencephalocele/Acephalostomia and Cleft lip; Anophthalmia and Microphthalmia; Spina Bifida/Holorachischisis. Major abnormalities of this combination and severity are rare in rats and the majority are outside of Historical Control Data range.

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

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

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

At 125 mg/kg/day there were 8 litters with similar major abnormalities, the majority affecting the head, eye and vertebral column. These abnormalities include, but are not limited to, Exencephaly/Meningoencephalocele/Acephalostomia and Cleft lip; Anophthalmia and Microphthalmia; Spina Bifida/Holorachischisis. Major abnormalities of this combination and severity are rare in rats and the majority are outside of Historical Control Data range.

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

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

The incidences of delayed ossification were within Historical Control Data range. This is an indication of fetal immaturity and as a transient stage in fetal development, not thought to be adverse.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

At 125 mg/kg/day there were 8 litters with similar major abnormalities, the majority affecting the head, eye and vertebral column. These abnormalities include, but are not limited to, Exencephaly/Me
ningoencephalocele/Acephalostomia and Cleft lip; Anophthalmia and Microphthalmia; Spina Bifida/Holorachischisis. Major abnormalities of this combination and severity are rare in rats and the majority are outside of Historical Control Data range.

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

At 125 mg/kg/day there were 8 litters with similar major abnormalities, the majority affecting the head, eye and vertebral column. These abnormalities include, but are not limited to, Exencephaly/Meningoencephalocele/Acephalostomia and Cleft lip; Anophthalmia and Microphthalmia; Spina Bifida/Holorachischisis. Major abnormalities of this combination and severity are rare in rats and the majority are outside of Historical Control Data range.

Dose descriptor:

LOAEL

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

visceral malformations

Remarks on result:

other: microphthalmia

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

external: eye

external: face

skeletal: skull

skeletal: vertebra

visceral/soft tissue: eye

Description (incidence and severity):

At 125 mg/kg/day there were 8 litters with similar major abnormalities, the majority affecting the head, eye and vertebral column. These abnormalities include, but are not limited to, Exencephaly/Meningoencephalocele/Acephalostomia and Cleft lip; Anophthalmia and Microphthalmia; Spina Bifida/Holorachischisis. Major abnormalities of this combination and severity are rare in rats and the
majority are outside of Historical Control Data range.
Also, at 125 mg/kg/day there was an increased incidence of medially thickened/kinked ribs, short
supernumerary cervical ribs, delayed ossification of 5th/6th sternebrae and thoracic/sacrocaudal vertebral elements and partially undescended lobe(s) of thymus compared to concurrent control.
The incidences of delayed ossification were within Historical Control Data range. This is an indication of fetal immaturity and as a transient stage in fetal development, not thought to be adverse.
At 30 mg/kg/day there was an incidence of Microphthalmia (not associated with severe cranial abnormalities). An incidence of 1 fetus in 1 litter is extremely low and is seen at this incidence within the control population as documented within the Historical Control Data range. However, as there were also 2 fetuses in 2 litters with Microphthalmia at 125 mg/kg/day as well as Anophthalmia, a relationship to treatment cannot be ruled out.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The results from the OECD414 pre-natal development study in rats indicate that Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives produces major skeletal abnormalities in the skull and spinal cord including effects in the eyes of microphthalmia and anophthalmia. The incidence of one fetus in one litter at 30 mg/kg/day is within the historical control range so 30mg/kg/day could be the true NOAEL, however a conservative NOAEL of 10mg/kg/day was selected as it was not possible to be sure that this effect was not treatment related.

Executive summary:

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

 

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

 

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

 

Results

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

 

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

 

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

 

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

 

Conclusion

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

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

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

The supporting substance CAS 71786 -60 -2 induced developmental toxicity (OECD 414)at 125 mg/kg/day in the absence of apparent maternal toxicity. But considering that clear maternal toxicity was found in the repeated dose toxicity studies (OECD 422 and OECD 408) at 125 mg/kg/day, H 361; Reprotox Cat 2 is considered to be appropriate.