Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

At this moment a Screening for reproductive/developmental toxicity (OECD 422) is being scheduled for the registered substance 2,2’-(C16-18 (even numbered) alkyl imino) diethanol ('HT-PFAEO') itself.Further there are several additional studies on reproduction toxicity available on comparable substances that only differ regarding the alkyl chain-length distributions. These studies have shown no effects on reproduction except for secondary to maternal toxicity at levels leading to significant mortality basically resulting from local gastro-intestinal toxicity rather than actual systemic toxicity. The most appropriate read across substance2,2'-(C16-18 (evennumbered, C18 unsaturated) alkyl imino) diethanol was tested in a combined repeated dose/reproduction toxicity screening (OECD 422) involving dosing by oral gavage for at least five weeks resulted to maternal and reproduction NOAEL of 75 mg/kg bw/day, due to mortalities during late gestation/early lactation and reduced offspring survival (low Day 4 and Day 13 lactation survival indices) at 175 mg/kg/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
19 August 2020 - 11 February 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH  
Primary Fatty Amine Ethoxylates (PFAEO) are substances derived from Primary Fatty Amines, ethoxylated with two moles of ethylene oxide to form a tertiary amine structure. The structure varies only with the length of the fatty amine alkyl chain length. The physico-chemical, fate, ecotoxicological and toxicological properties are expected to vary in a predictable pattern based only on the variation in chain length.
In this case the target substance 2,2’-(C16-18 (even numbered) alkyl imino) diethanol ('HT-PFAEO') varies from the source substance 2,2'-(C16-18 (evennumbered, C18 unsaturated) alkyl imino) diethanol ('Tallow_PFAEO') in that the source additionally contains C18-unsaturated alkyl chains. As consequence, both products are actually over 60% complete identical.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).
Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be considered as a category provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

The available data allows for an accurate hazard and risk assessment of the category, and the category concept is applied for the assessment of physicochemical properties, environmental fate and environmental and human health hazards. Thus, where applicable, environmental and human health effects are predicted from adequate and reliable data for source substance(s) within the group, by interpolation to the target substances in the group (read-across approach), applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006. In particular, for each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements for adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across.
The substances within the category of PFAEO are considered to apply to these general rules, and the similarity is justified on basis of scope of variability and overlapping of composition, representative molecular structure, physico-chemical properties, toxicological and ecotoxicological profiles and supported by various QSAR methods. There is convincing evidence that these chemicals lie in the overall common profile of this category or sub-category, respectively. The key points that the members share are:
a. Common origin: produced from Primary Fatty Amines, ethoxylated with two moles of ethylene oxide.
b. Similar structural features: aliphatic hydrocarbon chain bound to diethanol amine.
c. Similar physico-chemical properties: trend in log Pow based on alkyl chain length; low vapour pressure; water solubility decreasing with the alkyl chain length.
d. Common properties for environmental fate & eco-toxicological profile: readily biodegradable, no potential for bioaccumulation, comparable adsorption potential (independent to alkyl chain lengths), clear trend in aquatic toxicity (increasing toxicity with increasing carbon chain.
e. Similar chemical reactivity, absorption and metabolic pathways.
f. Common levels and mode of human health related effects
A detailed justification for the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Section 13).
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 29 July 2016
Deviations:
no
Remarks:
Some minor deviations from studyplan are reported which are considred no impact on the integrity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
RccHan™;WIST
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Envigo RMS Limited.
- Females (if applicable) nulliparous and non-pregnant: yes
- Weight at study initiation: Males 288 to 336 g; Females 194 to 229 g.
- Fasting period before study: no
- Housing: Limited barrier - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used throughout the study except during pairing. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily.
Distribution: The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.
Bedding: Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 day cycles were not allocated to the reproductive phase of the study.
Replacement before allocation Atypical estrous cycles: one female; Body weight range extremes: one female
Replacement during treatment: Welfare reasons: one female
DETAILS OF FOOD AND WATER QUALITY:
SDS VRF1 Certified pelleted diet. A sample (250 g) of each batch of diet used was retained within Pharmacy (frozen -10 to -30C) until finalization of the report. Samples were discarded after finalization of the report. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 20-24 deg.C
- Humidity (%): Monitored and maintained within the range of 40-70%.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark.

IN-LIFE DATES: From: 19 August 2020 (Arrival animals) To: 7 November 2020 (necropsy)
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was warmed until melted and gently inverted until homogenous. The required amount of test item was weighed. Approximately 50% of the final volume of vehicle was added and heated to around 60°C in a water bath whilst magnetically stirring until uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer and heated in water bath to around 60°C until homogenous. The formulation was transferred to the final containers, via syringe, whilst magnetically stirring.
The formulations were made in ascending order of concentration
Frequency of preparation: Twice weekly.
Storage of formulation: Refrigerated temperature (2 to 8°C).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not soluble in water.
- Concentration in vehicle: 0, 7.5, 18.75 and 43.75 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
Details on mating procedure:
- M/F ratio per cage:
Group 1 males : Group 1 females
Group 2 males : Group 2 females
Group 3 males : Group 3 females
Group 3 males* : Group 4 females
* Due to early termination of the Group 4 males, a second pairing of the Group 3 males with the Group 4 females will occur.
- Length of cohabitation: Up to 2 weeks.
- Proof of pregnancy: Ejected copulation plugs. Sperm within vaginal smear.
Day 0 of gestation When positive evidence of mating detected.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity: Homogeneity and stability of the test materials in the vehicle were determined as part of this program of work in Covance Study Number MH28JS. Formulations in the concentration range 1 to 100 mg/mL were stable at ambient temperature (15 to 25°C) for up to 1 day and at refrigerated temperature (2 to 8°C) for up to 4 days.
Achieved concentration: The first, fifth, ninth and last formulations prepared for administration were analyzed for achieved concentration of the test item.
Analysis: The method of analysis and results are reported
Duration of treatment / exposure:
Males 2 weeks before pairing up to necropsy after minimum of 5 weeks.
Females At least 2 weeks before pairing, then throughout pairing and gestation until Day 12 of lactation.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
175 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels for investigation have been selected in conjunction with the Sponsor and based on the results of a preliminary toxicity study Covance Study No. 8436494. In that study the systemic toxic potential of 2,2'-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol was assessed over a period of 14 days in Han Wistar rats at dose levels of 30, 75 and 175 mg/kg/day.
There were no premature deaths, no test item related changes in clinical condition and no effect of treatment on visual water intake. At 175 mg/kg/day piloerection was observed post-dose in all females on Day 5 and Day 7 of treatment. At the high dose level there was also an overall reduction in body weight gain observed in males and a reduction in food intake for males and females. Macroscopic findings; dark depressions (at 175 mg/kg/day) and dark areas (at 75 and 30 mg/kg/day) were observed on the stomachs of females, which were of unclear relationship to treatment. At 175 mg/kg/day increased body weight adjusted liver weights were observed in both sexes and a reduction in body weight adjusted spleen weights in males was also apparent.
The reduced body weight gain in males, reduced food intake in both sexes and macroscopic stomach findings in the females at 175 mg/kg/day were considered adequate to justify the choice of 175 mg/kg/day as the high dose on the current main OECD 422 study.
Intermediate and low dose levels are set at 75 and 30 mg/kg/day respectively in order to fulfill the 2-fold to 4- fold dosing interval as specified in the test guideline
- Fasting period before blood sampling for clinical biochemistry: No - Without overnight deprivation of food. Samples collected under light general anesthesia.
- Section schedule rationale (if not random): Not indicated.
Positive control:
Not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:

Minimum schedule • Week 1: daily.
• Week 2 to 4: twice weekly.
• Week 5 onwards: once each week.
• Gestation phase: Days 0, 7, 14 and 20.
• Lactation phase: Days 1, 6 and 12.

Detailed observations will be performed in the treatment period, at the following times during the day:
Dose observations • Pre-dose observation.
• 1 to 2 hours after completion of dosing.
• As late as possible in the working day.

BODY WEIGHT: Yes
- Time schedule for examinations:
Weekly during acclimatization.
Before dosing on the day that treatment commences and weekly thereafter.
On day of necropsy.
Females:
Days 0, 7, 14 and 20 after mating and
Days 1, 4, 7 and 13 of lactation.
More frequent weighing may be performed to aid the monitoring of the condition of animals displaying ill-health.

FOOD CONSUMPTION:
Weekly.
Food consumption will not be recorded for males and females during the period when paired for mating, but will recommence for each group of males once pairing of all the animals within a group is completed.
For females after mating food consumption schedule will match body weight schedule:
Days 0-7, 7-14, 14-20 after mating
Days 1-4, 4-7 and 7-13 of lactation.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Only visual water intake

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
0.5 mL in EDTA tubes with lithium heparin
- Time schedule for collection of blood:
At termination, from Sublingual vein.
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: No
- How many animals:
The five lowest numbered surviving males per group; The first five lactating females with a surviving litter per group
- Parameters checked in table were examined.

Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
•Prothrombin time (PT) - using IL PT Fibrinogen reagent.
•Activated partial thromboplastin time (APTT) - using IL APTT reagent.

CLINICAL CHEMISTRY: Yes
0.7 mL in
- Time schedule for collection of blood:
At termination, from Sublingual vein.
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: No
- How many animals:
The five lowest numbered surviving males per group; The first five lactating females with a surviving litter per group
- Parameters checked in table were examined.

PLASMA/SERUM HORMONES: Yes: T$ & TSH
- Animals fasted: No
Adult Terminal samples; 2 aliquots (tubes) per animal (one for T4 and one for TSH)
F0 Males: 40 per parameter (per animal - one sample/aliquot for T4 and one sample/aliquot for TSH – optional analysis – 80 in total)
F0 females (if relevant), at termination: 40 per parameter (per animal - one sample/aliquot for T4 and one sample/aliquot for TSH – optional analysis) – 80 in total
Serum samples obtained for thyroid stimulating hormone (TSH) analysis will be stored frozen (-60°C to -90°C) for up to 28 days (or longer; 3 months proven stability) as a contingency. Serum samples will be analyzed, if required. If the T4 investigations indicate treatment-related effects, then the relevance of further analysis for TSH will be discussed with the Sponsor.


Offspring:
Single aliquot per animal
Day 4 of age (if relevant) 40 per parameter, (per litter – one sample/aliquot for T4 and one sample/aliquot for TSH – optional analysis)- 80 in total (maximum)
Day 13 of age: 80 per parameter, (per sex, per litter – one sample/aliquot for T4 and one sample/aliquot for TSH – optional analysis) - 160 in total (maximum)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: sensory activity / grip strength / motor activity:
Sensory Reactivity and Grip Strength:
- Week 5 (before dosing) The five lowest numbered surviving males in each group
- Days 7-9 of lactation (before dosing): The first five lactating females in each group (length of separation of dam from litter must be minimized)
Motor Activity:
- Week 5 (before dosing) The five lowest numbered surviving males in each group
- Days 7-9 of lactation (before dosing): The first five lactating females in each group (length of separation of dam from litter must be minimized)

IMMUNOLOGY: No
Oestrous cyclicity (parental animals):
ESTROUS CYCLES:
Wet smears:
• For 14 days before treatment (all females including spares); animals that fail to exhibit 4-5 day cycles will not be allocated to study.
• After pairing until mating (for a maximum of 14 days).
• Females showing no evidence of mating
• For four days before scheduled termination (nominally Days 10 to 13 of lactation).
Dry smears:
•For at least 15 days before pairing, using cotton swabs.
Sperm parameters (parental animals):
For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD) (on day 1), pup weight on the day of AGD, presence of nipples/areolae in male pups,
Particular attention should be paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia.
T4 on Day 4 of age:
Offspring: up to 2 females per litter (where possible; reserve male pups for nipple retention evaluation):
• one for T4 (serum) (Priority given to T4 sample )
• one for TSH (serum)
No offspring will be allocated to these procedures on Day 4 of age if:
• the resultant live litter size would fall below 8 offspring
• the resultant number of female pups would fall below 3 offspring
• If only 4 female offspring are available within a litter but the overall litter size is >8, one female may be selected with priority given to the T4 sample.
Day 13 of age F1 offspring, 2 males and 2 females per litter (where possible)
• two for T4 (serum); where possible one male and one female (Priority given to T4 sample)
• two for TSH (serum); where possible one male and one female


GROSS EXAMINATION OF DEAD PUPS:
yes, Where possible fresh external macroscopic examination with an assessment of stomach for milk content. Grossly externally abnormal pups will be retained.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
Adult animals: Detailed macroscopic examination will be performed in an attempt to determine cause of ill health/death. Tissues retained as specified. Any abnormal tissues retained and may be weighed at the discretion of necropsy staff.
Offspring: Where possible fresh external macroscopic examination with an assessment of stomach for milk content. Grossly externally abnormal pups will be retained.
ORGAN WEIGHTS: Yes
For bilateral organs, left and right organs will be weighed together unless otherwise specified on the Pathology Procedures Table
Organ weights are not routinely recorded for animals killed or dying prematurely; organ weights will be recorded for groups terminated prematurely.

HISTOPATHOLOGY: Yes
Fixatives Standard : 10% Neutral Buffered Formalin.
Testes: Initially in modified Davidson’s Fluid.
Eyes: Davidson’s Fluid.
Processing - Full list All animals killed or dying prematurely.
The five lowest numbered surviving males in Groups 1 and 3 (being new HD group after early termination group 4) and the first five lactating females with a surviving litter in Groups 1 and 4 at scheduled termination.
Postmortem examinations (offspring):
Day 4 of age
Blood sampling required (see Thyroid hormone analysis Section).
Selected Day 4 offspring with no clinical observations discarded without examination.
Selected Day 4 offspring with clinical observations, examined externally, and retained pending possible future examination.

on Day 13 of age
Blood sampling required (see Thyroid hormone analysis Section).
Thyroid gland retained from one male and one female in each litter, where possible.
If insufficient numbers of Day 13 pups are available for hormone sampling and thyroid retention/preservation, priority is given to hormones, with sample for T4 given first priority.
All animals will be subject to an external macroscopic examination;
particular attention will be paid to the external genitalia.
Any abnormal tissues retained pending possible future examination.

Statistics:
Where appropriate, group mean values with standard deviation (SD), will be calculated from individual data.
For categorical data, the proportion of animals will be analyzed for each treated group (as appropriate) versus the control group.
For continuous data, Bartlett’s test will first be applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett’s test, treated groups will then be compared with the control group, incorporating adjustment for multiple comparisons where necessary.
The computer systems used may include those listed below:
Applied Biosystems/MDS Sciex Analyst: For data collection and processing, Bioanalysis (LC-MS/MS)
Liberate: In-house system used for reporting in-life, necropsy, pathology and statistics
Pristima: Pharmacy test item management, in-life, necropsy and pathology data collection
Quasar: In-house statistical analysis
Rodent Activity Monitoring System (RAMS): Activity monitoring
Sample Registry System: Dose Formulation Analysis data, Sample tracking
SAS: Statistical evaluation
StarTox: In-house statistical analysis
StatXact 3 statistical analysis package: Mating performance and fertility
Waters Empower: Dose Formulation Analysis, Bioanalysis, Pharmaceutical Analysis
Watson LIMS: Laboratory Information Management System for data quantification, Bioanalysis (LC-MS/MS)
Reproductive indices:
- Estrous cycles Individual animal values tabulated. Percentage of females showing the following cycle types calculated:
- Pre-coital interval Individual: intervals tabulated for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals calculated for durations of 1-4, 5-8, 9-12 and 13-14 days.
- Mating performance and fertility: Individual data tabulated. Group values calculated for males and females separately for the following: Percentage mating: Number animals mating x 100 / Animals paired
Conception rate: Number animals achieving pregnancy x 100 / Animals mated
Fertility index: Number animals achieving pregnancy x 100 / Animals paired
- Gestation length: Individual values tabulated for the number of days from mating to the start of parturition (inclusive), with half a day subtracted where parturition started overnight. Percentage of animals in appropriate categories tabulated for each group.
- Gestation index: Calculated for each group as: Number of live litters born x 100 / Number pregnant
- Litter size: Individual litter values tabulated for the number of implantation sites, total at Day 1 and live at Days 1, 4 and 13 of age.
Survival indices (%) Individual litter values calculated for:
- Post-implantation survival index : Total number offspring born x 100 / Total number uterine implantation sites
- Live birth index: Number live offspring on Day 1 after littering x 100 / Total number of offspring born
Offspring viability indices:
- Viability index: Number live offspring on Day 4 x 100 / Number live offspring on Day 1 after littering
- Lactation index: Number live offspring on Day 13 after littering x 100 /Number live offspring on Day 4 (after selection for thyroid hormone bleed)
Sex ratio: Individual litter values tabulated for total at Day 1 and live at Days 1, 4 (before and after selection for thyroid hormone bleed) and 13 of age.

Offspring examinations:
Individual values tabulated.
Ano-genital distance presented as distance in mm – may be normalized to a measure of pup size eg using body weight as a covariate.Nipple/areolae counts presented as mean numbers for males per litter and an overall group mean calculated.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The Group 4 males (175 mg/kg/day) were prematurely terminated on Day 9 of treatment. From this group; one male was found dead on Day 7, in addition two males on Day 7 and one male on Day 9 were euthanized for welfare reasons due to significant body weight loss and adverse clinical signs.
Clinical signs comprised; decreased activity, thin build conformation and piloerection in 3 males, as well as loose and mucoid feces in 2 males and hunched posture and ungroomed coat in 1 male.
Macroscopic examination revealed that all four animals had a small thymus, pale liquid within the organs of the gastrointestinal tract, with thickening of these organs also observed in the majority of cases. Dark areas or pale depressions on the stomach were also recorded in 3 of these males. For welfare reasons the remainder of the Group 4 males were prematurely terminated.
Several group 4 females were also euthanized for welfare reasons during gestation or early lactation (After having mated with Group 3 males after completion of mating with group 3 females), being in general poor clinical condition after showing signs of decreased activity, piloerection, hunch posture, pallor skin colour and abnormally cold to touch.
Mortality:
mortality observed, treatment-related
Description (incidence):
Treatment at 175 mg/kg/day was not tolerated; one male was found dead and three males were euthanized due to poor condition during the first nine days of the study, and three females were euthanized due to poor condition in late gestation/early lactation. The remaining males were euthanized on day 9 of the study to avoid any unnecessary suffering.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
body weight loss of -22g or -7g respectively. The body weight performance of males that received 75 mg/k/day subsequently improved, with body weight gain being similar to Controls for the remainder of the treatment period. As a result of the slight group mean body weight loss observed for males at 75 mg/kg/day, overall body weight gain (Days 1-36) was lower than Control. There was no effect of treatment on male body weight gain at 30 mg/kg/day. Group mean body weight gain for females that received 175 or 30 mg/kg/day was slightly low, compared to Controls, prior to pairing. At 175 mg/kg/day during gestation (Days 0-20), group mean body weight gain was low compared to Controls (30% lower) with statistical significance attained for the overall group mean body weight change.
On Day 1 of lactation, the group mean absolute body weight at 175 mg/kg/day was statistically significantly lower than Control, however, overall group mean body weight gain was higher than Controls during lactation (Day 1-13). There was no effect of treatment on body weight gain during gestation or lactation at 75 or 30 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was statistically significantly low for males that received 175 or 75 mg/kg/day during the first week of treatment (58% and 29% lower, respectively). Thereafter, food intake improved for males that received 75 mg/kg/day. At 30 mg/kg/day, male food consumption was unaffected by treatment. Food consumption for females that received 175 mg/kg/day was statistically significantly lower than Control prior to pairing and throughout gestation. Food intake for females at 175 mg/kg/day during lactation was only slightly lower than Control. There was no effect of treatment on female food consumption at 75 or 30 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Blood chemistry investigations at scheduled termination of the males revealed no treatment-related changes.
For males and, bile acids were increased in all groups of treated animals, however, statistical significance was not attained.
At scheduled termination of the females on Day 13 of lactation, blood chemistry investigations revealed a dose-related decrease in alkaline phosphatase (ALP), alanine transaminase (ALT) and aspartate aminotransferase (AST), with statistical significance attained at 175 mg/kg/day for ALP and at 175 and 75 mg/kg/day for ALT.
All other differences from Control, including those that attained statistical significance, were generally small, confined to one sex, or the magnitudes were not dose-related and, consequently, were considered to represent normal biological variation.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Blood chemistry investigations at scheduled termination of the males revealed no treatment-related changes.
For males and females, bile acids were increased in all groups of treated animals, however, statistical significance was not attained.
At scheduled termination of the females on Day 13 of lactation, blood chemistry investigations revealed a statistically significant dose-related decrease in alkaline phosphatase (ALP), alanine transa
minase (ALT) and aspartate aminotransferase (AST) at 175 mg/kg/day for ALP and at 175 and 75 mg/kg/day for ALT, which is of no biological significance
All other differences from Control, including those that attained statistical significance, were generally small, confined to one sex, or the magnitudes were not dose-related and, consequently, were considered to represent normal biological variation.
Endocrine findings:
no effects observed
Description (incidence and severity):
There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males. In the male and female offspring on Day 13 of age, T4 levels were slightly lower than Control at 175 mg/kg/day with statistical significance attained for the female offspring (Males and female values of treated groups were identical, also to F0 male, and not showing a dose-response relation. Only the control of the F1 females was a little higher than that of the males). There was no effect of treatment on the circulating levels of T4 in male or female offspring at 75 or 30 mg/kg/day. Due to lack of effects on T4, no TSH analysis have been performed.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no treatment-related clinical signs and no signs observed post-dose for males and females that received 75 or 30 mg/kg/day, and females that received 175 mg/kg/day during pre-mating period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related effects were observed for sensory reactivity and grip strength assessment during Week 5 of treatment for males at 75 or 30 mg/kg/day or during Days 7-9 of lactation for females.
Group mean forelimb and hindlimb grip strength values for males at 75 mg/kg/day and forelimb grip strength vales for females at 175 mg/kg/day were slightly low compared to Controls, however, these differences did not attain statistical significance and there was no dose-response.
Motor activity assessment of males at 75 or 30 mg/kg/day during Week 5 of treatment revealed no treatment-related effects.
Group mean high and low beam activity scores for males at 75 or 30 mg/kg/day showed some inter-group differences at the 54-minute time point, achieving a couple of isolated statistical significances, however, generally all other scores including the total scores were similar to Controls.
Motor activity assessment of females at 175 mg/kg/day during Days 7-9 of lactation revealed group mean low beam scores and to a lesser extent, high beam scores were slightly lower than Controls.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At scheduled terminal sacrifice, test item-related microscopic findings were noted in the gastrointestinal tract of males administered 75 mg/kg/day and of females administered 75 and 175 mg/kg/day.
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
At 175 mg/kg/day, three females showed an irregular cycle during treatment. All three females became normally pregnant and completed with litters.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
At 175 mg/kg/day, there was no effect of treatment on pre-coital interval, gestation length, conception rate or fertility index. Gestation index was slightly low for females that received 175 mg/kg/day, compared with Controls, with only 8 live litters born as one female was euthanized on Day 21 after mating.
There was no effect of treatment on estrous cycles, pre-coital interval, mating performance, fertility or gestation length at 75 or 30 mg/kg/day. At scheduled termination, all females were cycling and all were in diestrus.
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Remarks on result:
other:
Remarks:
The NOAEL is based on a just too severe toxicity at 175 mg/kg bw/day leading to significant mortaility and early termination of all males before mating, and several females at end pregancy/early lactation, although this seemed to be well tolerated during the RF. Despite the serious toxicity, affects on fertility seem limited. At 75 mg/kg/day there weer no effecets at reproduction.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
175 mg/kg bw/day (actual dose received)
System:
other: mortality, following local effects on gastro-intestinal system.
Organ:
intestine
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment at 175 mg/kg/day was associated with the following adverse signs in the offspring:
The offspring of female 4F 121 were cold to touch on Day 7 of age. In addition, the offspring of female 4F 122 were cold to touch with little/no milk in the stomach on Day 5 of age – with eight pups found dead or euthanized for welfare reasons. Furthermore, The offspring of female 4F 124 were cold to touch on Days 1 or 1-2 of age with the litter scattered in the cage on Days 1-2 and 11 pups found dead or euthanized.
There were no treatment-related clinical signs observed amongst the offspring at 75 or 30 mg/kg/day.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
As a result of a low post-implantation survival index at 175 mg/kg/day, total litter size was slightly lower than Control. At 175 mg/kg/day the viability index on Day 4 of lactation and lactation index on Day 13 were lower than Control.
At 75 and 30 mg/kg/day, litter size and survival indices were unaffected by maternal treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 175 and 75 mg/kg/day, it was noted that male and female offspring body weights were low on Day 1 of age, compared with Control.
At 175 mg/kg/day, overall (Day 1-13 of age) group mean body weight gain for male and female offspring was statistically significantly lower than Control. There was no effect of treatment on offspring body weight gain at 75 or 30 mg/kg/day.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Remarks on result:
other:
Remarks:
Due to the three mortalities during late gestation/early lactation and reduced offspring survival (low Day 4 and Day 13 lactation survival indices) at 175 mg/kg/day
Critical effects observed:
yes
Lowest effective dose / conc.:
175 mg/kg bw/day (nominal)
System:
other: Viability decreased, secondary to maternal toxicity/mortality
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
175 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
yes

As a result of a low post-implantation survival index at 175 mg/kg/day, total litter size was slightly lower than Control.  At 175 mg/kg/day the viability index on Day 4 of lactation and lactation index on Day 13 were also lower than Control and the extent of these changes were considered adverse.  At 75 or 30 mg/kg/day, litter size and survival indices were unaffected by maternal treatment.  Sex ratio was unaffected by treatment at all dose levels investigated.  There was no effect of treatment on ano-genital distance in the male and female offspring and the male offspring did not develop nipples.  Treatment at 175 mg/kg/day was associated with adverse clinical signs in the offspring including; offspring in three litters being recorded as cold to touch, with offspring in one of these litters having little/no milk in stomach and offspring in another litter being scattered in the cage.  Male and female offspring born to mothers treated at 175 and 75 mg/kg/day had low body weights on Day 1 of age, compared with Control.  At 175 mg/kg/day, overall (Day 1-13 of age) group mean body weight gain for male and female offspring was statistically significantly lower than Control, however, the extent of this change was considered non-adverse.  In the male and female offspring on Day 13 of age, T4 levels were slightly lower than Control at 175 mg/kg/day with statistical significance attained for the female offspring.

Conclusions:
Due to the three mortalities during late gestation/early lactation and reduced offspring survival (low Day 4 and Day 13 lactation survival indices) at 175 mg/kg/day, the NOAEL for reproductive/developmental toxicity was concluded to be 75 mg/kg/day.
Executive summary:

The purpose of this study was to assess the general systemic toxic potential of 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol in Han Wistar rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol by oral gavage administration for at least five weeks.

Three groups of ten male and ten female rats received 2,2'-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol at doses of 30, 75 or 175 mg/kg/day by oral gavage administration at a dose volume of 4 mL/kg/day. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. TheF1generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, Arachis oil, at the same volume dose as treated groups.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, thyroid hormone analysis and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

Results:

Treatment at 175 mg/kg/day was not tolerated: one male was found dead and three males were euthanized due to poor condition during the first nine days of the study, and three females were euthanized due to poor condition in late gestation/early lactation. The remaining males were euthanized on study day 9 to avoid any unnecessary suffering.

 

Toxicity parental generation (F0):

Test item-related macroscopic and microscopic findings were noted in the gastrointestinal tract of these early sacrificed male and female animals and intestinal lesions were considered the major factor contributing to death. Abnormalities were observed in the stomach, small and large intestine. Most of these animals presented with abnormal pale/dark/yellowish, gelatinous/thick content in the small and large intestine and rarely in the stomach. The test item-related findings in the smallintestine consisted of foamy macrophages expanding the lamina propria. Macroscopic lesions were graded up to moderate in severity likely causing intestinal malabsorption and impaired intestinal function. Hyperplastic and erosive lesions were seen in the stomach and were likely caused by contact of the test item with the nonglandular gastric mucosa which resulted in irritation, associated with adaptive changes in the epithelium. Necrosis, apoptosis and hyperplasia of the epithelium were observed in the large intestine. These adverse findings were also likely caused by contact of the test item with the mucosa, resulting in severe epithelial damage. Accumulation of foamy macrophages in the mesenteric lymph nodes were likely associated with impairment or delayed clearance of the test item and were considered non-adverse. Gastric hyperplasia and hyperkeratosis of the nonglandular epithelium were potentially the result of the irritant action of the test item on the gastric nonglandular mucosa causing adaptive epithelial modification at the cellular level and were therefore considered non‑adverse.

Due to the treatment-related mortality that occurred in four males and in three females administered 175 mg/kg/day, caused by intestinal lesions, this dose level was considered to exceed the maximum tolerated dose.

 

Effects reproduction and offspring (F1):

As a result of a low post-implantation survival index at 175 mg/kg/day, total litter size was slightly lower than Control. At 175 mg/kg/day the viability index on Day 4 of lactation and lactation index on Day 13 were also lower than Control and the extent of these changes were considered adverse. At 75 or 30 mg/kg/day, litter size and survival indices were unaffected by maternal treatment. Sex ratio was unaffected by treatment at all dose levels investigated. There was no effect of treatment on ano-genital distance in the male and female offspring and the male offspring did not develop nipples. Treatment at 175 mg/kg/day was associated with adverse clinical signs in the offspring including; offspring in three litters being recorded as cold to touch, with offspring in one of these litters having little/no milk in stomach and offspring in another litter being scattered in the cage. Male and female offspring born to mothers treated at 175 and 75 mg/kg/day had low body weights on Day 1 of age, compared with Control. At 175 mg/kg/day, overall (Day 1-13 of age) group mean body weight gain for male and female offspring was statistically significantly lower than Control, however, the extent of this change was considered non-adverse. In the male and female offspring on Day 13 of age, T4 levels were slightly lower than Control at 175 mg/kg/day with statistical significance attained for the female offspring.

Due to the three mortalities during late gestation/early lactation and reduced offspring survival (low Day 4 and Day 13 lactation survival indices) at 175 mg/kg/day, the NOAEL for reproductive/developmental toxicity was concluded to be 75 mg/kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
High quality study. Probably very conservative value as the next higher level of 175 mg/kg was just too high and resulted to parental mortality. This is supported by results from very comparable EOGRTS (not included in this dossier) with 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9, which resulted to a NOAEL reproduction of 150 mg/kg bw/day, the highest dose tested.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Read-across to Tallow-PFAEO:

Primary Fatty Amine Ethoxylates (PFAEO) are substances derived from Primary Fatty Amines, ethoxylated with two moles of ethylene oxide to form a tertiary amine structure. The structure varies only with the length of the fatty amine alkyl chain length. The physico-chemical, fate, ecotoxicological and toxicological properties are expected to vary in a predictable pattern based only on the variation in chain length.

The most appropriate read across substance for  2,2’-(C16-18 (even numbered) alkyl imino) diethanol ('HT-PFAEO') would be 2,2'-(C16-18 (evennumbered, C18 unsaturated) alkyl imino) diethanol  ('Tallow-PFAEO'). Target substance 'HT-PFAEO varies from the source substance Tallow-PFAEO in that the source additionally contains C18-unsaturated alkyl chains. As consequence, both products actually are over 60% complete identical.

Tallow-PFAEO was evaluated in a Combined repeated dose toxicity with a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints. Han Wistar rats were dose by oral gavage for at least five weeks at dose levels of 0, 30, 75 and 175 mg/kg/day.

Treatment at 175 mg/kg/day was not tolerated: one male was found dead and three males were euthanized due to poor condition during the first nine days of the study, and three females were euthanized due to poor condition in late gestation/early lactation. The remaining males were euthanized on study day 9 to avoid any unnecessary suffering. The toxicity was found to be related to local toxicity in the gastrointestinal tract.

As a result of a low post-implantation survival index at 175 mg/kg/day, total litter size was slightly lower than Control.  At 175 mg/kg/day the viability index on Day 4 of lactation and lactation index on Day 13 were also lower than Control and the extent of these changes were considered adverse.  At 75 or 30 mg/kg/day, litter size and survival indices were unaffected by maternal treatment.  Sex ratio was unaffected by treatment at all dose levels investigated.  There was no effect of treatment on ano-genital distance in the male and female offspring and the male offspring did not develop nipples.  Treatment at 175 mg/kg/day was associated with adverse clinical signs in the offspring including; offspring in three litters being recorded as cold to touch, with offspring in one of these litters having little/no milk in stomach and offspring in another litter being scattered in the cage.  Male and female offspring born to mothers treated at 175 and 75 mg/kg/day had low body weights on Day 1 of age, compared with Control.  At 175 mg/kg/day, overall (Day 1-13 of age) group mean body weight gain for male and female offspring was statistically significantly lower than Control, however, the extent of this change was considered non-adverse.  In the male and female offspring on Day 13 of age, T4 levels were slightly lower than Control at 175 mg/kg/day with statistical significance attained for the female offspring.

Due to the three mortalities during late gestation/early lactation and reduced offspring survival (low Day 4 and Day 13 lactation survival indices) at 175 mg/kg/day, the NOAEL for reproductive/developmental toxicity was concluded to be 75 mg/kg/day.

Additional information (not included in this dossier) involves a study on Oleyl-PFAEO, also a very comparable PFAEO, mainly consisting of C18-unsaturated alkyl chains. Oleyl-PFAEO was evaluated for effects on reproductive performance in an EOGRTS following ten weeks premating exposure duration for the parental (P0) generation.

This study concluded that the No Observed Adverse Effect Level (NOAEL) for the local irritant effect was not established and the NOAEL for both systemic and reproductive toxicity was considered to be 150 mg/kg/day.

A study (OECD 422) is planned to be performed on the HT-PFAEO substance itself in order to confirm acceptability of this read-across.

Effects on developmental toxicity

Description of key information

At this moment an OECD 414 pre-natal developmental toxicity study is being scheduled for the registered substance 2,2’-(C16-18 (even numbered) alkyl imino) diethanol itself.

Further there are several additional OECD 414 pre-natal developmental toxicity study available on comparable substances that only differ regarding the alkyl chain-length distributions. Themost appropriate read across substance 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9showed no indication of developmental toxicity in the foetuses, with the NOEL being 150 mg/kg.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
19th June 2013 to 17 the December 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH  

Primary Fatty Amine Ethoxylates (PFAEO) are substances derived from Primary Fatty Amines, ethoxylated with two moles of ethylene oxide to form a tertiary amine structure. The structure varies only with the length of the fatty amine alkyl chain length. The physico-chemical, fate, ecotoxicological and toxicological properties are expected to vary in a predictable pattern based only on the variation in chain length.
In this case the target substance 2,2'-(C16-18 (evennumbered, C18 unsaturated) alkyl imino) diethanol ('Tallow_PFAEO') varies from the source substance 2,2'-(octadec-9-enylimino)bisethanol ('Oleyl-PFAEO') in that the source mainly contains C18-unsaturated alkyl chains, whereas the target contains mostly a mix of C18:1 (most), C16 and C18 alkyl chains. As consequence, both products are for about 50% identical, and only some more C16 and C18 unsaturated are present in Tallow-PFAEO whereas this is C18:1 in Oleyl-PFAEO.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).
Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be considered as a category provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

The available data allows for an accurate hazard and risk assessment of the category, and the category concept is applied for the assessment of physicochemical properties, environmental fate and environmental and human health hazards. Thus, where applicable, environmental and human health effects are predicted from adequate and reliable data for source substance(s) within the group, by interpolation to the target substances in the group (read-across approach), applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006. In particular, for each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements for adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across.
The substances within the category of PFAEO are considered to apply to these general rules, and the similarity is justified on basis of scope of variability and overlapping of composition, representative molecular structure, physico-chemical properties, toxicological and ecotoxicological profiles and supported by various QSAR methods. There is convincing evidence that these chemicals lie in the overall common profile of this category or sub-category, respectively. The key points that the members share are:
a. Common origin: produced from Primary Fatty Amines, ethoxylated with two moles of ethylene oxide.
b. Similar structural features: aliphatic hydrocarbon chain bound to diethanol amine.
c. Similar physico-chemical properties: trend in log Pow based on alkyl chain length; low vapour pressure; water solubility decreasing with the alkyl chain length.
d. Common properties for environmental fate & eco-toxicological profile: readily biodegradable, no potential for bioaccumulation, comparable adsorption potential (independent to alkyl chain lengths), clear trend in aquatic toxicity (increasing toxicity with increasing carbon chain.
e. Similar chemical reactivity, absorption and metabolic pathways.
f. Common levels and mode of human health related effects
g. A detailed justification for the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Section 13).
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test”
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology Studies, 12 NohSan No 8147 (24 November 2000)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 190g to 269g.

The animals were housed individually in solid-floor polypropylene cages with stainless steel lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan UK, Oxon, UK) was used. Certificates of analysis of the batches of diet used are given in Appendix 14. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly mean temperatures and humidity were included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20% respectively; there were no deviations from these targets.

The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
As Arachis Oil was successfully used on both the twenty-eight day and ninety toxicity studies, the same vehicle and dosage (4 mL/kg body weight) was employed in this study. The stability and homogeneity of the test item formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services and showed the formulations to be stable for at least twenty one days at 4 °C. Formulations were therefore prepared in two separate bulk formulations (covering up to 9 days) and divided into daily aliquots and stored at approximately +4 °C in the dark.

Samples were taken of each test item formulation and were analyzed for concentration of 2,2'-(octadec-9-enylimino)bisethanol CAS No 25307-17-9 at Harlan Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within 94% to 104% of the nominal concentration and within acceptable limits of the nominal concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

Test Item
The test item described in the main part of this study was also used as the analytical standard.

Preparation of standard solutions
Stock solutins of test item in methanol were prepared for external standard calibration. An aliquot, 100 mg of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with methanol to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solutins were used to prepare working standard solutions in methanol with a concentration of 0.1 mg/mL.

Analysis of samples
The formulations recieved were extracted with methanol. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with methanol. This was then ultra-sonicated for 15 minutes and centrifuged to 4500 rpm for 10 minutes. Where necessary, sample solutions were further diluted with methanol to achieve the working concentration.

Preparation of accuracy samples
Samples of Arachis Oil BP were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These sample were then prepared for analysis.

Instrumental Setip

HC system: Agilent Technologues 5890, incorporating autosampler and workstation
Column: DB-1 (15 m x 0.53 mm id x 1.5 micro-m film)
Oven temperature program: Oven: 200°C for 0 minute, with 10°C/minute to 300°C, for 12 minutes
Injection temperature: 300°C
Flame ionisation detector temperature: 300°C
Injection volume: 1 micro-litre
Retention time: ~ 4.5 mins


Details on mating procedure:
Not described in the study
Duration of treatment / exposure:
Between Days 5 and 19 of gestation, inclusive.
Frequency of treatment:
Daily
Duration of test:
20 days
Remarks:
Doses / Concentrations:
15 mg/kg/day (3.75 mg/ml)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg/day (12.5 mg/ml)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg/day (37.5 mg/ml)
Basis:
actual ingested
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Justification
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Maternal examinations:
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. An additional observation was also performed five hours after dosing during the normal working week. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

Food Consumption
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

Water Consumption
Water intake was observed daily by visual inspection of the water bottles for any overt changes.
Ovaries and uterine content:
Post Mortem
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The ovaries and uteri of pregnant females were removed, examined and the following data recorded:

i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight

Implantation types were divided into:

Early Death: No visible distinction between placental/decidual tissue and embryonic tissue

Late Death: Separate embryonic/fetal and placental tissue visible

Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes

All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):


Left Horn Cervix Right Horn

L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16

V = viable fetus
Fetal examinations:
The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:

Body weight and body weight change (including adjustment for the contribution of the gravid uterus), food consumption, gravid uterus weight, litter data and fetal litter and placental weights: Bartlett’s test for homogeneity of variance. Where the data were shown to be homogeneous one way analysis of variance and, if significant, Dunnett’s multiple comparison test was employed, where the data were found to non homogeneous Kruskal-Wallis and, if significant, pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test was employed. Fetal evaluation parameters, including skeletal or visceral findings were analyzed by Kruskal-Wallis and, if significant, Mann-Whitney ‘U’ test.

Probability values (p) are presented as follows:

p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No effects of any toxicological significance
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter Responses
Litter Data and Litter Placental and Fetal Weights
There was no obvious effect of maternal treatment on the number of implantations, subsequent embryofetal survival and litter size, sex ratio and mean fetal, litter and placental weights on Day 20 of gestation at 15, 50 or 150 mg/kg bw/day.

At 150 mg/kg bw/day, mean pre-implantation loss was lower than control with differences attaining statistical significance. As animals were not dosed until implantation had occurred, these differences were incidental and unrelated to treatment.

At 15 and 50 mg/kg bw/day, higher mean female fetal weight and mean fetal weight attained statistical significance compared to control. In the absence of any similar increase in fetal weight at 150 mg/kg bw/day, this finding was considered to reflect normal biological variation and was unrelated to treatment.

Fetal Examination
Neither the type, incidence or distribution of findings observed externally at necropsy examination and subsequently during detailed visceral and skeletal assessment of the fetuses indicated any effect of treatment on fetal development.
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Mortality There were no unscheduled deaths during the study. Clinical Observations The low incidence of clinical sign observed during the study did not indicate any effect of treatment at 15, 50 or 150 mg/kg bw/day. Body Weight There was no effect of treatment on body weight or body weight gain, including when adjusted for the contribution of the gravid uterus, throughout the treatment period at 15, 50 or 150 mg/kg bw/day. Food Consumption There was no effect of treatment on food consumption throughout the treatment period at 15, 50 or 150 mg/kg bw/day. Water Consumption Daily visual inspection of water bottles did not reveal any overt intergroup differences. Post Mortem Studies No macroscopic abnormalities were detected for parental females at scheduled termination on Day 20 of gestation. Litter Responses Litter Data and Litter Placental and Fetal Weights There was no obvious effect of maternal treatment on the number of implantations, subsequent embryofetal survival and litter size, sex ratio and mean fetal, litter and placental weights on Day 20 of gestation at 15, 50 or 150 mg/kg bw/day. At 150 mg/kg bw/day, mean pre-implantation loss was lower than control with differences attaining statistical significance. As animals were not dosed until implantation had occurred, these differences were incidental and unrelated to treatment. At 15 and 50 mg/kg bw/day, higher mean female fetal weight and mean fetal weight attained statistical significance compared to control. In the absence of any similar increase in fetal weight at 150 mg/kg bw/day, this finding was considered to reflect normal biological variation and was unrelated to treatment. Fetal Examination Neither the type, incidence or distribution of findings observed externally at necropsy examination and subsequently during detailed visceral and skeletal assessment of the fetuses indicated any effect of treatment on fetal development.
Conclusions:
The No Observed Effect Level (NOEL) for the pregnant females and the survival, growth and embryofetal development of the offspring was considered to be 150 mg/kg bw/day.
Executive summary:

Introduction

The study was designed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.

 

The study was designed to comply with the following guidelines:

 ·        US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

·        Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

·        OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

·        Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

  

Methods

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD®(SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 15, 50, and 150 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Arachis oil) over the same treatment period to serve as a control. Clinical signs, body weight change, food and water consumptions were monitored during the study. 

 

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

 

Results….

Adult Responses

Mortality: There were no unscheduled deaths during the study.

Clinical Observations: Clinical sign did not indicate any effect of treatment at 15, 50 or 150 mg/kg bw/day.

Body Weight: Body weight and body weight gain, including adjustment for the contribution of the gravid uterus, was unaffected by treatment at 15, 50 or 150 mg/kg bw/day.

Food Consumption: Food consumption was unaffected by treatment at 15, 50 or 150 mg/kg bw/day.

Water Consumption: Daily visual inspection of water bottles did not reveal any overt intergroup differences.

Post Mortem Studies: No macroscopic abnormalities were detected for parental females at 15, 50 or 150 mg/kg bw/day.

Litter Responses

Litter Data and Litter Placental and Fetal Weights: The number of implantations, subsequent embryofetal survival and litter size, sex ratio and mean fetal, litter and placental weights on Day 20 of gestation were unaffected by maternal treatment at 15, 50 or 150 mg/kg bw/day.

Fetal Examination

There was no effect of maternal treatment on morphological development of the fetuses at 15, 50 or 150 mg/kg bw/day.

Conclusion

The No Observed Effect Level (NOEL) for the pregnant females and the survival, growth and embryofetal development of the offspring was considered to be 150 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
This study is high quality but based on results from very comparable EOGRTS with 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 which is the most appropriate read across available to for 2,2’-(C16-18 (even numbered) alkyl imino) diethanol. This study resulted to NOAEL reproduction of 150 mg/kg bw/day, the highest dose tested.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Read-across to Oleyl-PFAEO:

Primary Fatty Amine Ethoxylates (PFAEO) are substances derived from Primary Fatty Amines, ethoxylated with two moles of ethylene oxide to form a tertiary amine structure. The structure varies only with the length of the fatty amine alkyl chain length. The physico-chemical, fate, ecotoxicological and toxicological properties are expected to vary in a predictable pattern based only on the variation in chain length. The substance 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 ('Oleyl-PFAEO') is at the moment the most appropriate available read across substance for the read across on developmental toxicity.

The OECD 414 pre-natal development study on Oleyl-PFAEO included dose levels of 15, 50 and 150 mg/kg body weight, administered by gavage for days 5 to 19 of pregnancy as a solution in arachis oil. The dose levels were selected based on those used in the 28 day and 90 day repeat dose studies. The adult females did not show any signs of toxicity in this study such as on bodyweights, or food or water consumption, but there was no histopathological examination of their stomachs, making it not possible to see local adverse (irritant/corrosive) effects due to the corrosive/irritant nature of the test material as seen in the other repeat dose studies.

The absence of toxic effects in the adult females is not considered to be of concern as we have good evidence from the 28 day repeat dose study that higher doses could result in mortality or severe toxicity due to local effects in the stomach. 

The number of implantations, subsequent embryo/foetal survival and litter size, sex ratio and mean foetal, litter and placental weights on Day 20 of gestation were unaffected by maternal treatment at 15, 50 or 150 mg/kg bw/day.

There was no effect of maternal treatment on morphological development of the foetuses at 15, 50 or 150 mg/kg bw/day.

The No Observed Effect Level (NOEL) for the pregnant females and the survival, growth and embryo/foetal development of the offspring was considered to be 150 mg/kg bw/day. Oleyl-PFAEO it did not show any indications of potential for developmental toxicity in this study up to the maximum dose level of 150 mg/kg bw/day.

An additional OECD 414 pre-natal developmental toxicity study in rat is being scheduled for the registered substance 2,2’-(C16-18 (even numbered) alkyl imino) diethanol  (HT-PFAEO) itself in order to confirm acceptability of this read-across.

Mode of Action Analysis / Human Relevance Framework

Based on proposed MoA, grouping of cationic surface-active substances is possible (substances containing one or more long alkyl chains linked to a nitrogen). Under physiological conditions the nitrogen is protonated, and positively charged. This cationic surfactant structure leads to high adsorption at negatively charged surfaces such as cellular membranes. The cationic surfactant molecules then partition into the membranes in such a way that part of the molecule is associated to the polar head groups of the phospholipid while the non-polar alkyl chain partitions into hydrophobic environment of the membrane interior. A solute molecule partitioning in this way is, like the phospholipid molecules of which the membrane is composed, able to move freely in two dimensions only, but it cannot easily pass through the membrane to the intracellular environment. Noteworthy in this respect is that recent research shows that the log distribution coefficients for cationic surfactants between water and phospholipids are several orders of magnitude higher than between water and oil (Niels Timmer, Steven Droge, 2017). Consequently, the primary activity of cationic surfactants is disruption and leakage of cell membranes leading to damage or lysis of the cell content(s). Cytotoxicity through disruption of cell membrane at exposure site will occur rather than absorption over the cell membrane. Consequently, significant uptake followed by placental transfer is not expected to occur. This generic behaviour provides for a common mode of action for cationic surfactants that is not subject to species differences. Available data, including various OECD 414 in rabbit, on all these substances combined however, indicate that these effects do not lead to, or are linked to, effects on reproduction or development.

The available data from available repeated dose studies and reproduction toxicity studies shows that dosing is limited because of local effects in gastro-intestinal system, specifically in the stomach. Further, no adverse effects on reproductive organs were identified in the 28 and 90-day studies in rats. And specifically in the developmental toxicity and EOGRTS there were no indications of possible reproductive toxicity.

Justification for classification or non-classification

The most relevant studies available for 2,2’-(C16-18 (even numbered) alkyl imino) diethanol do not give rise to specific concerns for effects on fertility or foetal development. Consequently, there is no need for classification for effects on reproduction.

Additional information