2,2'-(C16-18 (evennumbered) alkyl imino) diethanol

Endpoint summary

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.

Any further need for the performance of an EOGRTS on 2,2’-(C16-18 (even numbered) alkyl imino) diethanol ('HT-PFAEO') itself will be revaluated upon completion of the OECD 422 and OECD 414 on the substance.

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

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

Primary reasoning:

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. The degree of saturated to unsaturated C18 alkyl chains represents the constituents with the largest degree of variation between these two products. As consequence, both products actually are over >60% identical.

The category approach was undertaken to identify an appropriate read-across substance for 2,2’-(C16-18 evennumbered alkyl imino) diethanol CAS No 1218787-30-4 based on sufficient data density and k-nearest neighbor structural identity that could be considered a reasonably conservative approach. 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6 is the output of this approach. Lower alkyl chain-lengths are available in the category such as 2,2’-(C12-18 evennumbered alkyl imino) diethanol, (PFAEO-C), 71786-60-2 which demonstrate increased levels of toxicity. It is theorized that decreasing alkyl chain length on this family of tertiary amines leads to increased toxicity among several endpoints.

While 2,2’-(C12-18 evennumbered alkyl imino) diethanol CAS No 71786-60-2 is the most toxic category member, 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6 is the most reasonable member for comparison purposes at it has the highest degree of similarity while offering a conservative trend approach to 2,2’-(C16-18 evennumbered alkyl imino) diethanol CAS No 1218787-30-4. It was for this reason and the other reasons listed above that PFAEO-T was choosen as the read-across substance for PFAEO-HT.

 

Read-across to Tallow-PFAEO:

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.  Despite the serious toxicity, affects on fertility seem limited with only post-implantation index being slightly lower than control which was an effect of the maternal animals sacrificed in extremis in late gestation. 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.

 

Information on addtional category members:

Addtionally, Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9) has shown to affect reproductive parameters in rats. The oral administration of PFAEO-C to rats by gavage, at dose levels of 125, 30 and 10 mg/kg/day, resulted in treatment related effects at 125 and 30 mg/kg/day. These effects consisted of a localized irritant effect, with an almost complete regression observed following the treatment-free period. Due to the inevitable variability in the counting of corpora lutea in females four days after littering, ranges observed inside that of historical controls, and the lack of a dose response, these differences are not considered to be evidence of a toxic effect on reproduction. 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. There is currently a EOGRTS being performed on PFAEO-C that will help to better define the nature of this toxicity.

 

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.

The available study on 2, 2’-(Octadec-9-enylimino) bisethanol is an  TG OECD 443 extended one generation reproductive toxicity study. There were observed reductions in body weight gain and food consumption in both the F0 and F1 generations as well as increases in some hematological parameters. These effects were determined to be secondary to the gastrointestinal inflammation and irritancy observed at all doses. The extent of the local irritancy of the findings in addition to deaths in the high dose group (150 mg/kg/day) resulted in an overall NOAEL being set to 30 mg/kg/day. Litter size, survival indices and sex ratio the number of implantation sites, post-implantation survival, live birth index and the number of offspring on Day 1 (total and live Day 1) were unaffected by parental treatment at dose levels up to and including 150 mg/kg/day. Resulting in the conclusion systemic and reproductive toxicity was determined to have a NOAEL of 150 mg/kg/day. This study concluded that the No Observed Adverse Effect Level (NOAEL) for the local irritant effect was not established with the LOAEL of 30 mg/kg bw /day.

 

On-going work:

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

Reference 1

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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

19 August 2021 - 03 December 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See read-across documentation in section 13.1

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: • Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

RccHan®:WIST. (Han Wistar) rat

Details on test animals or test system and environmental conditions:

Strain/Species RccHan®:WIST. (Han Wistar) rat.
Supplier Envigo RMS Ltd. (UK)
Number of animals ordered 88 time-mated females.
Duration of acclimatization Four days from arrival on Day 2 after mating to commencement of treatment on Day 6 after mating.
Age of the animals at the start of the study (Gestation Day 2) 11 weeks old.
Weight range of the animals at the start of the study (Gestation Day 3) 171 to 238 g.

3.3.2 Mating
Method Natural mating with Han Wistar males at the supplier’s facility. Males and females were not related.
Day 0 of gestation When positive evidence of mating was detected.
Delivery to Labcorp On Day 2 after mating

3.3.3 Allocation and Identification
Allocation Randomly to each group, on day of arrival.
Method Allocation was controlled to prevent time mated females mated with the same male partner being allocated to the same 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.

3.4 Animal Care and Husbandry

3.4.1 Environmental Control
Animal 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 20-24C 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.

3.4.2 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.
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, which was changed at appropriate intervals each week.
Number of animals per cage One.

3.4.3 Environmental Enrichment
Aspen wood based products A soft white untreated wood block: provided to each cage throughout the study and replaced when necessary.
Plastic shelter Provided to each cage throughout the study and replaced at the same time as the cages.

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

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

3.4.6 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 wood based products.
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:

Route Oral gavage using a suitably graduated syringe and a flexible cannula inserted via the mouth.

Method For each group, the required amount of dose was drawn up into the syringe and cannula. The outside of the cannula was wiped clean of any formulation residue with a clean disposable tissue and the end of the flexible cannula was lightly tapped onto clean tissue to remove any remaining droplets. The cannula was then dipped into a container filled with 5% glucose solution to aid intubation.

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 20 (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.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Correction factor None.

Vehicle Arachis oil

Method of preparation 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.

Frequency of preparation Twice weekly.

Storage of formulation Refrigerated (2 to 8C).

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.


Stability and homogeneity The homogeneity and stability of formulations during storage were determined as part Labcorp Study Number MH28JS. In that study, formulations in the range 1 to 100 mg/mL were determined to be stable for:
• One day at ambient temperature (15 to 25C)
• Four days when stored refrigerated (2 to 8C)

Achieved concentration Samples of each formulation prepared for administration in the first and last weeks of treatment were analyzed for achieved concentration of the test item.

Analysis The method of analysis and results are presented in Attachment 14.2.

Details on mating procedure:

Method Natural mating with Han Wistar males at the supplier’s facility. Males and females were not related.

Day 0 of gestation When positive evidence of mating was detected.

Delivery to Labcorp On Day 2 after mating

Duration of treatment / exposure:

Females were treated from Day 6 to Day 20 (inclusive) after mating, once daily at approximately the same time each day.

Volume dose 4 mL/kg body weight.

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

Frequency of treatment:

Once daily

Duration of test:

14 days Day 6 to Day 20 post-coitum

Dose / conc.:

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

Remarks:

Control

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

22 Females

Control animals:

yes, concurrent vehicle

Maternal examinations:

3.6.1 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. 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 during the treatment period at the following times in relation to dose administration:
• Pre-dose observation
• 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 3, 5, 12, 18 and 21 after mating to monitor general health.

3.6.2 Body Weight
The weight of each adult was recorded on Days 3 and 6-21 after mating.

3.6.3 Food Consumption
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 3-5, 6-8, 9-11, 12-14, 15-17 and 18-20 after mating inclusive.

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 21 after mating.

Organ Weights
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin

3.7.3 Histology
Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

Thyroid All adult females.

Routine staining Sections were stained with hematoxylin and eosin.

3.7.4 Light Microscopy

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 (Salewski, 1964)).

Blood sampling:

3.6.4 Thyroid Hormone Analysis
Blood samples were collected at the following occasion GD 21 at necropsy

Parameters
Triiodothyronine (T3)
Thyroxine (T4)
Thyroid stimulating hormone (TSH)

Sequence of blood sampling on each occasion To minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was controlled to allow satisfactory inter-group comparisons. Animals were sampled in a random order, within a two-hour time period, where possible

Conditions No overnight deprivation of food.

Blood sample site Sublingual vein.

Anesthetic Isoflurane.

Anticoagulant None.

Blood tubes Greiner Minicollect tubes with clotting activator.

Blood volume 1.0 mL.

Treatment of samples Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation.

Centrifugation conditions At 2000g for ten minutes at 4°C.
Serum was transferred to appropriately labelled polypropylene “cryo” tubes using plastic disposable pipettes and then mixed by gentle ten-fold inversion. Following mixing, each serum sample was divided in two aliquots.

Number of aliquots Two per animal.
Aliquot 1: 0.2 mL serum for T3/T4
Aliquot 2: residual serum for TSH

Final storage conditions Deep frozen (approximately -60°C to -90C) pending analysis.

Fate of samples Aliquot 1 (T3 and T4): dispatched to the department of LC MS/MS Bioanalysis, Labcorp.
Aliquot 2: dispatched to the Department of Immunology and Immunotoxicology, Labcorp.

T3 and T4 Performed by the department of LC-MS/MS Bioanalysis, Labcorp.
The method of analysis and results are presented in Attachment 14.3.

TSH Performed by the Department of Immunology and Immunotoxicology, Labcorp.
The method of analysis and results are presented in Attachment 14.4.

Fetal examinations:

3.7.6 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. Fetuses examined externally with abnormalities recorded. Particular attention was paid to the external genital organs of male fetuses. The sex and ano-genital distance of each fetus was recorded.

50% of fetuses in each litter Sexed internally, eviscerated and fixed in Industrial Methylated Spirit (IMS).

50% of fetuses in each litter Sexed internally and examined for visceral abnormalities by fresh microdissection (Modified Staples technique) and subsequently fixed in Bouin’s solution.

Processing Bouin’s fixed fetuses had heads removed post-fixation and heads were processed by Wilsons free-hand serial sectioning. Torsos were retained in Bouin’s solution.
IMS fixed fetuses were processed and stained with Alizarin Red.

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

Alizarin Red stained fetuses Assessed for skeletal development and abnormalities.

Statistics:

This report only contains serial observations pertaining to the gestation period (from Day 3 after mating).
Where appropriate, group mean values, each with standard deviation (SD), were calculated from individual data. Summary tabulated data was normally restricted to data derived from females/litters with live young at Day 21 after mating.

For all adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
The following data types were analyzed at each timepoint separately:
Body weight, using absolute weights 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)
Placental, litter and fetal weights
Ano-genital distance, average for each litter adjusted for litter average fetal body weight
Organ weights, absolute

The following comparisons were performed:
Group 1 vs 2, 3 and 4

The following sequence of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, pre/post implantation loss, live young, sex ratio - percentage male, placental, litter and fetal weights, ano-genital distance and organ weight data

For litter average ano genital distance, analysis of covariance was performed using average fetal weight for each litter as covariate (Angervall and Carlstrom, 1963), unless non parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in fetal weight which might influence the ano genital distance.

Indices:

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:

Available and used for comparison where necessary and to help validate controls.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The appearance and behavior of all animals were unaffected by treatment.

Isolated findings of decreased activity, salivation and piloerection were reported in one animal receiving 150 mg/kg/day, but in view of the low incidence, this was considered unrelated to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Overall body weight gain (Day 6-21 after mating) was statistically significantly lower than control in females receiving 150 mg/kg/day (overall reduction of 50%). The extent of the reduction from control was greatest from Day 13-21 after mating (55% reduction, compared to a 41% reduction from Day 6-13 after mating).

Body weight gain was unaffected in females receiving 15 or 50 mg/kg/day.

On Day 21 of gestation, gravid uterine weight was statistically significantly lower than control in females receiving 150 mg/kg/day (overall reduction 28%). When adjusted for the contribution of the gravid uterus, there was a mean loss of body weight (-6 g) in females receiving 150 mg/kg/day.

Gravid uterus weight and adjusted body weight gain was unaffected in females receiving 15 or 50 mg/kg/day.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Overall mean food intake during the dosing period (Day 6-21 after mating) was 24% lower than controls in females receiving 150 mg/kg/day. There was no similar finding at 15 or 50 mg/kg/day.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

The analysis of thyroid hormone concentrations on Day 21 of gestation revealed statistically significantly low triiodothyronine concentration in females receiving 150 mg/kg/day, compared with control.
Thyroxine concentration was slightly lower than control in females receiving 50 or 150 mg/kg/day and thyroid stimulating concentration was slightly high in females receiving 150 mg/kg/day but in each case, there was no dose response and statistical significance was not attained and, as such, this was not attributable to treatment.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no organ weight differences considered to be related to treatment with 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol in the thyroid and parathyroid glands.
Any changes in the absolute thyroid and parathyroid weights of treated females were considered to be incidental and not related to treatment, as there was no microscopic correlate. It is possible that any changes in the absolute thyroid and parathyroid weights of females receiving 150 mg/kg/day were secondary to lower than control bodyweight gain and lower than control food intake seen in females receiving 150 mg/kg/day, and not related to treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no macroscopic findings considered to be related to treatment with 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol.
The thyroids of one female treated with 150 mg/kg/day were macroscopically small, and this finding was considered to represent normal biological variation as there was no microscopic correlate.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item related microscopic findings were seen in the thyroid gland.

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The number of live offspring was slightly low and pre-and post-implantation loss were slightly high at 150 mg/kg/day, with the mean number of live offspring and pre- and post-implantation losses being outside of the historical control range. However, the pre-implantation loss, although outside the range of the historical control data, was only marginally higher than that at 50 mg/kg/day, did not attained statistical significance and represents a period prior to the start of dosing. The number of live offspring and post-implantation losses did not attain statistical significance and individual values were similar to control. These variations were therefore not attributable to treatment

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

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):

At scheduled termination on Day 21 after mating, two control females (Nos. 14 and 17), three females receiving 15 mg/kg/day (Nos. 34, 35 and 41), one female receiving 50 mg/kg/day (No. 57) and four females receiving 150 mg/kg/day (Nos. 71, 72, 80 and 82) were not pregnant. In addition, one female receiving 15 mg/kg/day (No. 25), one female receiving 50 mg/kg/day (No. 45) and one female receiving 150 mg/kg/day (No. 79) had a total litter resorption

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Total litter weight was 29% lower than control at 150 mg/kg/day, the difference attaining statistical significance. There was no effect on total litter weight for females receiving 15 or 50 mg/kg/day.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Total litter weight was 29% lower than control at 150 mg/kg/day, the difference attaining statistical significance. There was no effect on total litter weight for females receiving 15 or 50 mg/kg/day.

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

Placental weight was slightly higher than control in females receiving 150 mg/kg/day. There was no effect of maternal treatment on male, female or overall fetal weight at any dose level investigated

Key result

Dose descriptor:

NOAEL

Effect level:

> 150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

Treatment related:

no

Summary of Thyroid Hormone Levels after 13 Weeks of Treatment

Group/sex	1F	2F	3F	4F
Dose level (mg/kg/day)	0	15	50	150
Thyroxine (pg/mL)	19500	21300	17700	16000
Triiodothyronine (pg/mL)	493	515	494	354**
Thyroid stimulating hormone (pg/mL)	580	725	596	741

** p≤0.01

Conclusions:

It is concluded that that oral administration of 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol to Han Wistar rats during the organogenesis and fetal growth phases of pregnancy at doses of 0, 15, 50 or 150 mg/kg/day was generally tolerated but cause an adverse reduction of weight gain, associated with low food intake, in females receiving 150 mg/kg/day. The number of implantations, resorptions and live young, the pre- and post implantation loss and sex ratio were considered unaffected by treatment. The No-Observed-Adverse-Effect Level (NOAEL) for maternal toxicity was therefore considered to be 50 mg/kg/day, based on the effects on body weight gain and body weight loss when corrected for gravid uterus. The NOAEL for embryo-fetal survival and development was considered to be 150 mg/kg/day.

Executive summary:

The purpose of this study was to assess the influence of 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Han Wistar rat. Dose levels of 0, 15, 50, 150 mg/kg bw/day. The dose levels for this study were selected based on the results of Labcorp Study Number 8466317. In that study, a dose of 150 mg/kg/day was tolerated by pregnant Han Wistar rats, but resulted in a reduction of weight gain (associated with low food intake), which, when adjusted for gravid uterus weight, resulted in an overall body weight loss. There was no effect on embryo-fetal survival or development. The lower body weight gain was considered not to preclude the use of the 150 mg/kg/day in this main study, which was, therefore, selected as the highest dose.

The purpose of this study was to assess the influence of 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Han Wistar rat. This study demonstrated that doses up to 150 mg/kg/day were generally tolerated with no treatment-related clinical signs and no premature deaths, though there was evidence of a toxic response in pregnant females which was manifest as an adverse reduction of body weight gain (50% reduction from control), adjusted weight loss and a sustained reduction in food consumption. There was no treatment-related effect on the number of implantations, resorptions and live young, the pre- and post implantation loss and sex ratio and there were no treatment-related findings at the fetal pathology investigations, indicating that there was no effect on embryo-fetal development. Although there was a statistically significant decrease of triiodothyronine concentration in females receiving 150 mg/kg/day, there was considered to be no effect of treatment on the hormonal control of the thyroid gland, since thyroxine and thyroid stimulating hormone concentrations were unaffected and there was no effect on thyroid gland weight and pathology.

Fetal showed bodyweight reductions of up to 29% in the high dose group when compared with that of control. This effect was not considered adverse. Addtionally, secondary effects of maternal toxicity at 150 mg/kg bw/day  resulting in an average body weight loss of 50% when corrected for gracid uterine weight as this may have contributed to reduced fetal bodyweight. 

The reproductive assessment revealed high pre- and post-implantation loss in females receiving 150 mg/kg/day, resulting in a lower number of implantations and live young, which was adverse, with all values being outside of the background control range. The reduction in live young correlated with lower total litter weights and higher placental weights at this dose level. There was, however, no effect on individual fetal weights and the increased placental weights were likely due to the lower numbers of live young in each litter.

Maternal NOAEL was set to 50 mg/kg bw/day due to severe effects on body weight gain.

Fetal NOAEL is 150 mg/kg/day due to lack of adverse effects seen.

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

General reasoning:

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 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol CAS No 1218787-32-6 ('PFAEO T') is at the moment the most appropriate available read across substance for the read across on developmental toxicity.

There is no current data for developmental toxicity on 2,2’-(C16-18 evennumbered alkyl imino) diethanol CAS No 1218787-30-4 though there is a current OECD 414 in progress. The category approach was undertaken to identify an appropriate read-across substance for 2,2’-(C16-18 evennumbered alkyl imino) diethanol CAS No 1218787-30-4 based on sufficient data density and k-nearest neighbor structural identity that could be considered a reasonably conservative approach. 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6 is the output of this approach. Lower alkyl chain-lengths are available in the category such as 2,2’-(C12-18 evennumbered alkyl imino) diethanol, (PFAEO-C), 71786-60-2 which demonstrate increased levels of toxicity. It is theorized that decreasing alkyl chain length on this family of tertiary amines leads to increased toxicity among several endpoints.

 

Read-across to Oleyl-PFAEO:

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.

 

Read-across to PFAEO T:

An OECD 414 was also conducted on 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol CAS No 1218787-32-6, which has an average shorter alkyl chain-length (C16-18). It is concluded that that oral administration of 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol to Han Wistar rats during the organogenesis and fetal growth phases of pregnancy at doses of 0, 15, 50 or 150 mg/kg/day was generally tolerated but cause an adverse reduction of weight gain, associated with low food intake, in maternal females receiving 150 mg/kg/day. There was an adverse increase of pre- and post-implantation loss and a lower number of live young and smaller litter size at 150 mg/kg/day. This could possibly be a secondary effect of maternal local toxicity and bodyweight lose, though a direct test item related effect cannot be ruled out. The No-Observed-Adverse-Effect Level (NOAEL) for maternal toxicity and embryo-fetal survival and development was therefore considered to be 50 mg/kg/day. 

An OECD 422 on 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol did have lower live birth indexes though this is thought to be a factor of the reduced post-implatation indexes. Further lower foetal bodyweights, PND 4 survival and PND 13 surival were all decreased at 150 mg/kg bw/day compared to that of controls. This factors are reduced as three maternal females were sacrificed in late gestation /early lactation. This leads the authors of the study to the conclusion that 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) had no effect on implantation count and live birth index. Reduced offspring survival (low Day 4 and Day 13 lactation survival indices) resulted in a conservative NOAEL of 75 mg/kg bw/day. Mortalities in maternal animals resulted in a NOAEL of 75 mg/kg bw/day for repeat-dose toxicity.

 

Addtional category information:

2,2’-(C12-18 evennumbered alkyl imino) diethanol CAS No 71786-60-2
has shown in particular to affect developmental and reproductive parameters in both rats and some developmental parameters in rabbits. 

The results from the OECD 414 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.

The results from the OECD 414 pre-natal development study in rabbits indicate that Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives showed treatment related toxicity at the high dose of 175 mg/kg/day causing overt maternal toxicity which manifested as body weight loss and low food consumption (one female was killed following a sustained period of low/negligible food intake and associated clinical signs), however, embryo fetal survival was unaffected but mean fetal weights were slightly low and there was an increase in incomplete ossification, a transient, non-adverse minor fetal finding. It was concluded from this study that the dosage of 60 mg/kg/day is the maternal no observed-adverse-effect-level (NOAEL), and the no observed adverse-effect-level (NOAEL) for embryo-fetal survival, growth and development is 60 mg/kg/day.

There is currently a EOGRTS being performed on PFAEO-C that will help to better define the nature of this toxicity.

 

On-going work

An addtional OECD 414 is being conducted currently on 2,2’-(C16-18 evennumbered alkyl imino) diethanol CAS No 1218787-30-4. While no conclusions can yet be made on the study itself, the conclusion of the study will help to solidify conclusions and trends in the category. 

 

Conclusion

There is a clear break in the trends of developmental toxicity for the category of PFAEOs. This break occurs somewhere between C12 and C16 alkyl chain lengths. The avialable information on the category points to increasing chain lengths and increasing saturation resulting in decreased toxicity. 2,2’-(C16-18 evennumbered alkyl imino) diethanol CAS No 1218787-30-4 is close to the edge of teh break in this category in terms of chain-length. As there is no estabished trend for the endpoint of developmental toxcity in terms of degrees of severity leading to predictable outcomes at the lower boarder of chain lengths. It is prudent to examine category members with the closest structural relationship in order to determine risk and hazard while still ensuring a conservative approach. The substance 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6 provides the best canidate for this approach as it has a lower degree of saturation and a slightly higher proportion w/w% of C16 chain-lengths, thus establishing a conservative approach while maintaining a high degree of similarity to 2,2’-(C16-18 evennumbered alkyl imino) diethanol CAS No 1218787-30-4.

 

Justification for selection of Effect on developmental toxicity: via oral route:

We have a Klimisch 1 full GLP compliant OECD414 study on 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6 an appropriate read across substance for  2,2’-(C16-18 evennumbered alkyl imino) diethanol CAS No 1218787-30-4. However this OECD414 study does not cover the implantation period, which is covered by the OECD 422 on the same substance. Addtional information is provided by the OECD 443 on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9.

 

Justification for selection of Effect on developmental toxicity: via inhalation route:

Due to the low vapour pressure and the physical form of the substance, it being a waxy paste a room temperature inhalation is not an expected route of exposure in the workplace.  In addition the irritant properties to the skin would make such testing very technically difficult.

 

Justification for selection of Effect on developmental toxicity: via dermal route:

The skin irritant properties of this substance mean the repeated dose dermal studies are not scientifically justified due to concerns for animal welfare.  Also t would be expected to be absorbed less through the skin than orally, therefore it is possible to calculate dermal DNELs based on the available oral repeat dose NOAEL data, based on ECHA guidance.

 

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.

General reasoning

There is no specific test data on potential reproductive toxic effect of for 2,2’-(C16-18 evennumbered alkyl imino) diethanol CAS No 1218787-30-4 though there is an OECD 422 is currently in progress. An appropriate read across substance 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6 was determined appropriate to read-across for developmental toxicity endpoints based of comparable structures. 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6, is predominantly C16-18 with approximately 50% of the C18 unsaturated, while, 2,2’-(C16-18 evennumbered alkyl imino) diethanol CAS No 1218787-30-4 is predominantly C16-18 saturated. The unsaturated C18 is more reactive and therefore expected to be more toxic than the equivalent C18 saturated structure. Addtionally, 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6 has slightly more C16 constituents on average than it counterpart. The decrease in saturation and alkyl cahin length is thought to be more toxic based on trend in the available data, constituting a conservative trend approach worse-case scenario.

Sufficent data density and structural similarity exist on the proposed category for the given endpoints of reproductive and developmental toxicity that would allow reasonable conclusions to be drawn. Available trends within the data of the category allow for assertion that lower alkyl-chain lengths led to increasing severity of adversity of foetal toxic effects. Addtionally, these comounds that are proposed for read-across share high degrees of similarity assessed via the tanimoto index. As stated above, within the trends established in this category approach2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6 a worse-case than in regard to reproductive and developmental toxicity.

 

Specific reasoning to Reproductive classification

The OECD 422 that is available for 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6 demonstrated that treatment at 175 mg/kg bw/day exceeded the tolerable intake level of the animals and resulted in the mortality of four male rats and an addtional three females that were in late gestation/early lactation.  One female at 175 mg/kg/day and one female at 30 mg/kg/day failed to litter and were confirmed to not be pregnant at necropsy. Implantation count and live birth index were unaffected by maternal treatment with 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6. As a result of a low post-implantation survival index at 175 mg/kg/day, total litter size was slightly lower than Control, though this is an effect of maternal mortality in late gestation. At 175 mg/kg/day the viability index on Day 4 of lactation and lactation index on Day 13 were lower than Control as a result of the maternal mortalites. At 75 or 30 mg/kg/day, litter size and survival indices were unaffected by maternal treatment.

These results indicate that there is little concern for reproductive toxicity for 2,2’-(C16-18 evennumbered alkyl imino) diethanol CAS No 1218787-30-4 as it has a increased level of saturation and increased chain length which both show trends of decreasing concerns toxicity among reproductive endpoints.

 

Specific reasoning to Developmental classification

There is a break in the category approach for PFAEOs for developmental toxcity that lies somewhere between category members 2,2’-(C12-18 evennumbered alkyl imino) diethanol CAS No 71786-60-2 (primarily C12 alkyl chain) and 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6 (primarily C16-18 alkyl chain). Results from OECD 414 on 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6 do not show the same foetal malformations observed in the OECD 414  on 2,2’-(C12-18 evennumbered alkyl imino) diethanol CAS No 71786-60-2 at comparable dose levels, high-dose 125 and 150 mg/kg bw/day respectively. 

This information presents with two distinct posibilities either a.) there is a break in developmental toxicity between C12 and C16 alkyl chain lengths for this endpoint, or b.) overt maternal toxicity at the highest dose level tested (150 mg/kg bw/day) for 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6 thus any higher dosing structure to elicit similar responses would be too detrimental to the welfare of maternal animals to evaluate the response. Regardless, it is clear that 2,2’-(C12-18 evennumbered alkyl imino) diethanol CAS No 71786-60-2 is not a clear indicator of developmental toxicity for other category members with alkyl chains that exceed C16. Severe maternal toxicity from corrosive effects driven by the lipophilic nature of the molecule seems to take precedent in this category at chain lengths of C16-18 and does not result in developmental malformations observed with their C12 counterparts at the tolerable doses tested.

 "In principle, adverse effects on reproduction seen only at very high
dose levels in animal studies (for example doses that induce prostration, severe inappetence, excessive mortality) would not normally
lead to classification,..." Section 3.7.2.5.8. EC 1272/2008

Obsereved trends in the data among available OECD 414s in the category suggest that maternal toxicity decreases with increasing chain length. The OECD 414 on 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6 did show high pre- and post-implantation loss in females receiving 150 mg/kg/day, resulting in a lower number of implantations and live young. The reduction in live young correlated with lower total litter weights and higher placental weights at this dose level. There was, however, no effect on individual fetal weights and the increased placental weights were likely due to the lower numbers of live young in each litter.  Addtionally, secondary effects of maternal toxicity at 150 mg/kg bw/day  resulting in an average body weight loss of of maternal animals by 50% when corrected for gravid uterine weight as this may have contributed to reduced fetal bodyweight and acted a as a possible precursour for implantation loss due to the poor health of the maternal animals. These effects on reproductive and developmental parameters were not observed at lower dose levels that did not induce maternal bodyweight loss.

 

Conclusions

The OECD414 pre-natal development study in rats, showed no evidence of developmental toxicity even at the top dose of 150mg/kg bodyweight, therefore while this study does not include dosing during the pre-mating period until day 4, it does not indicate any potential for developmental toxicity (adverse effects on the morphological development of the foetus). The combine evidence from the OECD414 and OECD422 studies indicates that 2,2’-(Octadecylimino)bisethanol can be considered not to induce adverse effects on the morphological development of the foetus in rats or exhibit direct effects on fertility in either sex, therefore it is not necessary to classify this substance for reproductive or developmental toxicity based on current data from read-across to 2,2’-(C16-18 evennumbered alkyl imino) diethanol CAS No 1218787-30-4 .

Based on available informaiton on the category where the substance 2,2’-(C16-18 evennumbered alkyl imino) diethanol CAS No 1218787-30-4,  lies in the domain of the category with respect to chain length, the apperent breaks in toxicity, and reproductive/developmental effects only occuring at doses that induce maternally toxic effects resulting in bodyweight loss, it is not considered necessary to categorize 2,2’-(C16-18 evennumbered alkyl imino) diethanol CAS No 1218787-30-4 based on available infromation for developmental or reproductive toxicity.

To confirm this conclusion OECD 422 and OECD 414 tests are currently being conducted on 2,2’-(C16-18 evennumbered alkyl imino) diethanol CAS No 1218787-30-4. These results will help to establish possible trends or breaks in toxicity at lower category boundary in regard to chain-length.