Octadecan-1-ol, ethoxylated, phosphates

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and also for reproductive/developmental toxicity was considered to be 1000 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 October 2018 to 26 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Purity: 100% (UVCB)
Appearance/Physical state: Solid, white pastille
Storage: At ambient temperature (15 to 25°C)

Species:

rat

Strain:

other: RccHan™:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain/Species: RccHan™:WIST rat.
Supplier: Envigo RMS UK.
Number of animals ordered: 44 males and 48 females.

Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization
Males: six days before commencement of treatment.
Females: 20 days before commencement of treatment.

Age of the animals at the start of treatment
Males: 84 to 90 days old.
Females: 98 to 104 days old.

Weight range of the animals at the start of treatment
Males: 314 to 358 g.
Females: 191 to 236 g.

Allocation and Identification

Allocation
On arrival and non-selective allocation to cages.

Estrous cycles were evaluated pre-treatment.

After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study.

On Day 1 of study all animals were weighed and body weights were reviewed by Study Management before dosing commenced to ensure variations in body weight of animals did not exceed +/- 20% of the mean for each sex.

Identification of animals
Each adult animal was assigned a number and identified uniquely within the study by microchip before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.

Identification of cages
Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Animal Replacement
Before the commencement of treatment, any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.

Replacement before allocation
Abnormal estrous cycles in three females.

Animal Care and Husbandry

Environmental Control

Animal facility
Limited barrier - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.

Air supply
Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity
Monitored and maintained within the range of 20-24ºC and 40-70%.

There were no deviations from these ranges.

Lighting
Artificial lighting, 12 hours light : 12 hours dark.

Electricity supply
Public supply with automatic stand-by generators.

Animal Accommodation

Cages
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.

Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods.

Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.

Cage 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.

Number of animals per cage:
Pre-pairing - up to four animals of one sex
Pairing - one male and one female
Males after mating - up to four animals
Gestation - one female
Lactation - one female + litter

Environmental Enrichment

Aspen chew block
A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.

Plastic shelter
Provided to each cage throughout the study (except during pairing and from Day 20 after mating) and replaced at the same time as the cages.

Nesting material
Paper shavings were provided from Day 20 after mating and were changed at the same frequency as the bedding.

Diet Supply

Diet
SDS VRF1 Certified pelleted diet.

A sample (250g) of each batch of diet used was retained within Pharmacy (frozen, -10 to -30 deg.C) until finalization of the report. The sample was discarded after finalization of the report.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Method of preparation

Dosing was restricted to the F0 generation

The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer.

A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.

Frequency of preparation
Weekly.

Storage of formulation
Ambient temperature (15 to 25 deg.C).

Details on mating procedure:

Pairing commenced: After a minimum of two weeks of treatment.
Male/female ratio: 1:1 from within the same treatment groups.
Duration of pairing: Up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 10 and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. The stability was confirmed following storage at ambient temperature (15-25 deg.C) for up to 15 days, and for up to 15 days refrigerated (2-8 deg.C).

Achieved concentration
Samples of each formulation prepared for administration in Week 1 and the final week of treatment were analyzed for achieved concentration of the test item.

Duration of treatment / exposure:

Males
Two weeks pre-pairing up to necropsy after minimum of five weeks.

Females
Two weeks before pairing, then throughout pairing and gestation until Day 12 of lactation.

Frequency of treatment:

Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.

Details on study schedule:

F0 males: After final investigations completed (after at least five weeks of treatment).
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females: Day 13 of lactation.
F1 offspring: Selected offspring for thyroid hormone analysis - Day 4 of age.
Scheduled kill - Day 13 of age.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

330 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose selection was based upon a range-finding study - see Repeat dose toxicity/supporting study/Envigo 2019.

Parental animals: Observations and examinations:

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

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

Signs Associated with Dosing
Detailed observations were performed on F0 generation animals to establish and confirm a pattern of signs in association with dosing according to the following schedule:

F0 males
Week 1 - daily
Week 2 onwards - weekly

F0 females
Week 1 - daily
Week 2 - once

Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 12

Detailed observations were recorded 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

Detailed physical examination and arena observations
Before treatment commenced, and during each week of treatment, and on Days 0, 6, 13 and 20 after mating, and Days 1, 6 and 12 of lactation, detailed physical examinations and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities. “Blind” recording was not possible for animals during pairing or for females after mating and during lactation, for logistical reasons, therefore, observations were made on these occasions without “blinding”.

After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.

Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

Sensory reactivity and grip strength
Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the first five lactating females in each group at Days 7-9 of lactation. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, male cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. For females, animals were removed and returned to their home cages by an assistant, such that the observer was unaware of the treatment group. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups to the extentas far as possible on each day of testing.

Motor activity
During Week 5 of treatment for males and at Days 7-9 of lactation for females, the motor activity of the five lowest numbered surviving males and the first five lactating females in each group was measured (before dosing) using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.

Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not all necessarily tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.

Body Weight
The weight of animals was recorded as follows:

F0 males
Weekly during acclimatization
Before dosing on the day that treatment commenced and weekly thereafter.
On the day of necropsy.

F0 females
Weekly during acclimatization
Before dosing on the day that treatment commenced and weekly before pairing.
Days 0, 7, 14 and 20 after mating.
Day 1, 4, 7 and 13 of lactation.
On the day of necropsy.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:

F0 animals
Weekly, from the day that treatment commenced.

Food consumption was not recorded for males and females during the period when paired for mating, but recommenced for males once pairing for all animals were completed.

For females after mating food consumption was performed to match the body weight recording:

Days 0-6, 7-13 and 14-19 after mating
Days 1-3, 4-6 and 7-12 of lactation.

From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

Oestrous cyclicity (parental animals):

Dry and wet smears were taken as follows:
Dry smears For 15 days before pairing using cotton swabs
Wet smears Using pipette lavage during the following phases:
- For 14 days before treatment (all females including spares); animals that fail to exhibit 4-5 day cycles were not be allocated to study.
- After pairing until mating (for a maximum of 14 days).
- For four days before scheduled termination (nominally Days 11 to 14 of lactation).

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:

Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter: Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights: Days 1, 4, 7 and 13 of age.
Day 1 - all F1 offspring.
Day 13 of age - male offspring.

Postmortem examinations (parental animals):

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.

Time of Necropsy
F0 males: After final investigations completed (after at least five weeks of treatment).
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females#; Day 13 of lactation.

Postmortem examinations (offspring):

F1 offspring Selected offspring for thyroid hormone analysis - Day 4 of age.
Scheduled kill - Day 13 of age.

Statistics:

Statistical analyses were performed on the majority of data presented, whether significant or non-significant. For some parameters, including estrous cycles before treatment and stage of estrous cycle at termination, the similarity of the data was such that analyses were not considered to be necessary.

All statistical analyses were carried out separately for males and females. Data relating to food consumption were analyzed on a cage basis. For all other 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.

Reproductive indices:

The following were calculated for each litter:

Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

Group mean values were calculated from individual litter values.

Offspring viability indices:

The following were calculated for each litter:

Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100

Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) / Number live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 13 after littering / Number of live offspring on Day 4 (after blood sampling)) x 100

Group mean values were calculated from individual litter values.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Sensory reactivity observations was similar for animals treated at 100, 330 or 1000 mg/kg/day, when compared with the Controls. For males and females receiving 1000 mg/kg/day, the hindimb grip strength was slightly but statistically significantly lower than the Controls. In the absence of a similar effect on forelimb grip strength or a dose response in females, this finding is considered unlikely to be related to treatment: further, all values were within the historical control data range so no effect of treatment was inferred.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weight and body weight gain for males during Days 1-36 of treatment was similar to Controls across all groups. The body weight gain of males receiving 1000 mg/kg/day was statistically significantly lower than Control from Days 29-36 of treatment. In the absence of any similar effect on food consumption, and in view of the absence of an effect on overall body weight gain, this is considered incidental in these adult animals and not related to treatment.

Mean body weight and body weight gain for females during Days 1-15 of treatment (before pairing) was similar to Controls across all groups.

The body weight gain of females receiving 330 or 1000 mg/kg/day was slightly lower than that of the Control during Gestation Days 14-20, resulting in statistically significantly lower overall body weight gain during gestation. This is considered a result of the slightly lower litter sizes in these groups, and not related to treatment. The bodyweight gain of females receiving 100 mg/kg/day was similar to that of the Control throughout gestation.

The bodyweight gain of females receiving 100, 330 or 1000 mg/kg/day was higher than that of the Controls during Days 1-4 of lactation; a dose response was not apparent. The body weight gain of these females thereafter was generally similar to that of the Control during Days 4-13 of lactation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption of all animals was considered unaffected by treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological investigations in Week 6 for males and on Day 13 of lactation for females did not reveal any findings that were considered related to treatment.
The investigations in Week 6 for males revealed slightly but statistically higher white blood cell counts in males receiving 100, 330 or 1000 mg/kg/day when compared with the Control, although a dose response was not apparent. In addition, lymphocyte counts were slightly but statistically significantly higher in males receiving 100 mg/kg/day when compared with Controls, eosinophil counts were slightly but statistically higher than Control for males receiving 100, 330 or 1000 mg/kg/day and activated partial prothrombin times were slightly but statistically shorter than Control for males receiving 1000 mg/kg/day. These differences did not show a dose response, so no effect of treatment was inferred.

The investigations on Day 13 of lactation for females receiving 330 or 1000 mg/kg/day revealed slightly but statistically higher neutrophil counts when compared with the Control. This difference did not show a dose response, so no effect of treatment was inferred.

All other differences were minor, confined to one sex or did not show a relationship to treatment and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Biochemical examination of blood plasma in Week 6 for males and on Day 13 of lactation for females did not reveal any findings that were considered related to treatment.

The investigations in Week 6 for males revealed slightly lower, but statistically significant low creatinine concentrations when compared with Control in males receiving 100, 330 or 1000 mg/kg/day, and statistically significantly higher phosphorus concentrations in males receiving 100 mg/kg/day.

The biochemical examination of the blood plasma on Day 13 of lactation for females revealed, when compared with the Controls, statistically significantly lower A/G ratios in females receiving 1000 mg/kg/day. This difference occurred in isolation, so no effect of treatment was inferred.

All other differences were minor, confined to one sex or did not show a relationship to treatment and were therefore attributed to normal biological variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The clinical condition of the animals, their behavior in the arena, sensory reactivity, grip strength and motor activity were all unaffected by treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Change related to treatment with octadecan-l-ol, ethoxylated, phosphates was seen in the jejunum.

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of various cell types present within the different stages. No cell or stage specific abnormalities were noted. The qualitative examination of the ovary also revealed no abnormality.

Jejunum
Lymphangiectasis were seen in all males and some females given 1000 mg/kg/day.

All other microscopic findings were considered incidental and unrelated to test item.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in offspring on Day 13 of age. There was therefore no requirement to measure T4 in the samples obtained from offspring on Day 4 of age or from the adult females and none of the TSH (thyroid stimulating hormone) samples required analysis.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females allocated to study showed normal 4/5 day estrous cycles during the acclimatization period.

Estrous cyclicity, pre-coital interval, fertility, mating performance, gestation length and index were unaffected by treatment.

All females were not cycling before termination (Days 11 to 14 of lactation) and were in diestrous at termination.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Unaffected by treatment.

Reproductive performance:

no effects observed

Description (incidence and severity):

Unaffected by treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects

Key result

Reproductive effects observed:

no

F1 Litter Responses

The clinical condition of the offspring, offspring survival and sex ratio were unaffected by parental treatment. Litter size and offspring body weight on Day 1 of age were unaffected by treatment; offspring weight gain was statistically significantly higher at 100, 330 and 1000 mg/kg/day. This is considered to be related to the slightly lower litter sizes in these groups and not an effect of treatment with the test item.

Ano-genital distance of both male and female offspring on Day 1 of age and male nipple counts on Day 13 of age showed no effects of parental treatment.

Macroscopic examination of offspring that died prior to the scheduled termination or were killed on Day 13 of age did not reveal any findings that were considered related to parental treatment at any dose level.

Conclusions:

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and also for reproductive/developmental toxicity was considered to be 1000 mg/kg/day.

Executive summary:

The purpose of this study was the assessment of systemic toxic potential in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item, octadecan-1-ol, ethoxylated, phosphates, by oral gavage administration for at least five weeks.

Three groups of ten male and ten female rats received octadecan-1-ol, ethoxylated, phosphates at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration. 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. The F1 generation 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, corn 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, estrous cycles, pre-coital interval, mating performance, fertility, gestation length,thyroid hormone analysis,organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age. Blood samples were collected from selected offspring on Day 4 (not analyzed) and Day 13 of age for thyroid hormone analysis. 

Results

The mean concentrations of Octadecan-1-ol, ethoxylated, phosphates (CAS RN 62362-49-6) in test formulations analyzed for the study were within +10/-15% of nominal concentrations, confirming accurate formulation. The difference from mean remained within 5%, confirming precise analysis, with the exception of Week 4 Group 3, which was +/- 5.06%.

Parental (F0) responses

There wer no premature deaths related to treatment with the test item. 

Clinical condition, behavior in the arena, sensory reactivity, grip strength and motor activity were unaffected by treatment. There were no signs seen in association with dosing.

The slight variations of mean body weights and body weight gains in females during the dosing period were considered likely to be related to litter size and not treatment with the test item.

The food consumption of all animals was considered unaffected by treatment.

Estrous cycles, pre-coital interval, fertility, mating performance, gestation length and index were unaffected by treatment. There was no effect of treatment on the number of implantations or litter size.

Haematological investigations of the plasma and biochemical examinations of the blood did not reveal any findings that could be attributed to treatment.

There was no effect of treatment on the circulating levels of thyroxine (T₄) in adult males or in the Day 13 offspring.

The evaluation of organ weights of males after 5 weeks of treatment and of females on Day 13 of lactation revealed no significant differences when compared with the Controls.

The macroscopic and microscopic examination of adult males and females revealed lymphangiectasis in the jejunum of all males and some females receiving 1000 mg/kg/day. There was no change in the jejunum of animals receiving 100 or 330 mg/kg/day.

F1 Litter Responses

The clinical condition of the offspring, offspring survival and sex ratio were unaffected by parental treatment. Litter size and offspring body weight on Day 1 of age were unaffected by treatment; offspring weight gain was statistically significantly higher at 100, 330 and 1000 mg/kg/day. This is considered to be related to the slightly lower litter sizes in these groups and not an effect of treatment with the test item.

Ano-genital distance of both male and female offspring on Day 1 of age and male nipple counts on Day 13 of age showed no effects of parental treatment.

Macroscopic examination of offspring that died prior to the scheduled termination or were killed on Day 13 of age did not reveal any findings that were considered related to parental treatment at any dose level.

Conclusion

In conclusion, oral administration of the test item, Octadecan-1-ol, ethoxylated, phosphates (CAS RN 62362-49-6), to parental Han Wistar rats at dose levels of 100, 330 or 1000 mg/kg/day for five weeks to males and for two weeks before pairing, throughout gestation and up to Day 13 of lactation in females was well-tolerated in the adult animals with no treatment related adverse effects observed. 

Reproductive performance, fertility and offspring survival were unaffected by parental treatment. There was no effect of treatment on the number of implantations, litter size or the growth of the offspring.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and also for reproductive/developmental toxicity was considered to be 1000 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 October 2018 to 26 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Purity: 100% (UVCB)
Appearance/Physical state: Solid, white pastille
Storage: At ambient temperature (15 to 25°C)

Species:

rat

Strain:

other: RccHan™:WIST

Details on test animals or test system and environmental conditions:

Strain/Species: RccHan™:WIST rat.
Supplier: Envigo RMS UK.
Number of animals ordered: 44 males and 48 females.

Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization
Males: six days before commencement of treatment.
Females: 20 days before commencement of treatment.

Age of the animals at the start of treatment
Males: 84 to 90 days old.
Females: 98 to 104 days old.

Weight range of the animals at the start of treatment
Males: 314 to 358 g.
Females: 191 to 236 g.

Allocation and Identification

Allocation
On arrival and non-selective allocation to cages.

Estrous cycles were evaluated pre-treatment.

After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study.

On Day 1 of study all animals were weighed and body weights were reviewed by Study Management before dosing commenced to ensure variations in body weight of animals did not exceed +/- 20% of the mean for each sex.

Identification of animals
Each adult animal was assigned a number and identified uniquely within the study by microchip before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.

Identification of cages
Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Animal Replacement
Before the commencement of treatment, any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.

Replacement before allocation
Abnormal estrous cycles in three females.

Animal Care and Husbandry

Environmental Control

Animal facility
Limited barrier - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.

Air supply
Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity
Monitored and maintained within the range of 20-24ºC and 40-70%.

There were no deviations from these ranges.

Lighting
Artificial lighting, 12 hours light : 12 hours dark.

Electricity supply
Public supply with automatic stand-by generators.

Animal Accommodation

Cages
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.

Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods.

Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.

Cage 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.

Number of animals per cage:
Pre-pairing - up to four animals of one sex
Pairing - one male and one female
Males after mating - up to four animals
Gestation - one female
Lactation - one female + litter

Environmental Enrichment

Aspen chew block
A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.

Plastic shelter
Provided to each cage throughout the study (except during pairing and from Day 20 after mating) and replaced at the same time as the cages.

Nesting material
Paper shavings were provided from Day 20 after mating and were changed at the same frequency as the bedding.

Diet Supply

Diet
SDS VRF1 Certified pelleted diet.

A sample (250g) of each batch of diet used was retained within Pharmacy (frozen, -10 to -30 deg.C) until finalization of the report. The sample was discarded after finalization of the report.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Method of preparation

Dosing was restricted to the F0 generation

The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer.

A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.

Frequency of preparation
Weekly.

Storage of formulation
Ambient temperature (15 to 25 deg.C).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 10 and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. The stability was confirmed following storage at ambient temperature (15-25 deg.C) for up to 15 days, and for up to 15 days refrigerated (2-8 deg.C).

Achieved concentration
Samples of each formulation prepared for administration in Week 1 and the final week of treatment were analyzed for achieved concentration of the test item.

Details on mating procedure:

Pairing commenced: After a minimum of two weeks of treatment.
Male/female ratio: 1:1 from within the same treatment groups.
Duration of pairing: Up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Duration of treatment / exposure:

Males
Two weeks pre-pairing up to necropsy after minimum of five weeks.

Females
Two weeks before pairing, then throughout pairing and gestation until Day 12 of lactation.

Frequency of treatment:

Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

330 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose selection was based upon a range-finding study - see Repeat dose toxicity/supporting study/Envigo 2019.

Maternal examinations:

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

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

Signs Associated with Dosing
Detailed observations were performed on F0 generation animals to establish and confirm a pattern of signs in association with dosing according to the following schedule:

F0 females
Week 1 - daily
Week 2 - once

Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 12

Detailed observations were recorded 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

Detailed physical examination and arena observations
Before treatment commenced, and during each week of treatment, and on Days 0, 6, 13 and 20 after mating, and Days 1, 6 and 12 of lactation, detailed physical examinations and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities. “Blind” recording was not possible for animals during pairing or for females after mating and during lactation, for logistical reasons, therefore, observations were made on these occasions without “blinding”.

After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.

Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

Sensory reactivity and grip strength
Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the first five lactating females in each group at Days 7-9 of lactation. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, male cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. For females, animals were removed and returned to their home cages by an assistant, such that the observer was unaware of the treatment group. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups to the extentas far as possible on each day of testing.

Motor activity
During Week 5 of treatment for males and at Days 7-9 of lactation for females, the motor activity of the five lowest numbered surviving males and the first five lactating females in each group was measured (before dosing) using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.

Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not all necessarily tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.

Body Weight
The weight of animals was recorded as follows:

F0 males
Weekly during acclimatization
Before dosing on the day that treatment commenced and weekly thereafter.
On the day of necropsy.

F0 females
Weekly during acclimatization
Before dosing on the day that treatment commenced and weekly before pairing.
Days 0, 7, 14 and 20 after mating.
Day 1, 4, 7 and 13 of lactation.
On the day of necropsy.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:

F0 animals
Weekly, from the day that treatment commenced.

Food consumption was not recorded for males and females during the period when paired for mating, but recommenced for males once pairing for all animals were completed.

For females after mating food consumption was performed to match the body weight recording:

Days 0-6, 7-13 and 14-19 after mating
Days 1-3, 4-6 and 7-12 of lactation.

From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

Ovaries and uterine content:

For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.

Number of implantation sites was counted and confirmed if none were visible at visual inspection for non-pregnant females.

Fetal examinations:

Premature deaths
Where possible, a fresh macroscopic examination (external) with an assessment of stomach for milk content was performed. Abnormal tissues were retained.

Statistics:

Statistical analyses were performed on the majority of data presented, whether significant or non-significant. For some parameters, including estrous cycles before treatment and stage of estrous cycle at termination, the similarity of the data was such that analyses were not considered to be necessary.

All statistical analyses were carried out separately for males and females. Data relating to food consumption were analyzed on a cage basis. For all other 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.

Indices:

The following were calculated for each litter:

Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100

Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) / Number live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 13 after littering / Number of live offspring on Day 4 (after blood sampling)) x 100

Group mean values were calculated from individual litter values.

Historical control data:

Historical control data was presented in the report.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Sensory reactivity observations was similar for animals treated at 100, 330 or 1000 mg/kg/day, when compared with the Controls. For males and females receiving 1000 mg/kg/day, the hindimb grip strength was slightly but statistically significantly lower than the Controls. In the absence of a similar effect on forelimb grip strength or a dose response in females, this finding is considered unlikely to be related to treatment: further, all values were within the historical control data range so no effect of treatment was inferred.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weight and body weight gain for males during Days 1-36 of treatment was similar to Controls across all groups. The body weight gain of males receiving 1000 mg/kg/day was statistically significantly lower than Control from Days 29-36 of treatment. In the absence of any similar effect on food consumption, and in view of the absence of an effect on overall body weight gain, this is considered incidental in these adult animals and not related to treatment.

Mean body weight and body weight gain for females during Days 1-15 of treatment (before pairing) was similar to Controls across all groups.

The body weight gain of females receiving 330 or 1000 mg/kg/day was slightly lower than that of the Control during Gestation Days 14-20, resulting in statistically significantly lower overall body weight gain during gestation. This is considered a result of the slightly lower litter sizes in these groups, and not related to treatment. The bodyweight gain of females receiving 100 mg/kg/day was similar to that of the Control throughout gestation.

The bodyweight gain of females receiving 100, 330 or 1000 mg/kg/day was higher than that of the Controls during Days 1-4 of lactation; a dose response was not apparent. The body weight gain of these females thereafter was generally similar to that of the Control during Days 4-13 of lactation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption of all animals was considered unaffected by treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological investigations in Week 6 for males and on Day 13 of lactation for females did not reveal any findings that were considered related to treatment.
The investigations in Week 6 for males revealed slightly but statistically higher white blood cell counts in males receiving 100, 330 or 1000 mg/kg/day when compared with the Control, although a dose response was not apparent. In addition, lymphocyte counts were slightly but statistically significantly higher in males receiving 100 mg/kg/day when compared with Controls, eosinophil counts were slightly but statistically higher than Control for males receiving 100, 330 or 1000 mg/kg/day and activated partial prothrombin times were slightly but statistically shorter than Control for males receiving 1000 mg/kg/day. These differences did not show a dose response, so no effect of treatment was inferred.

The investigations on Day 13 of lactation for females receiving 330 or 1000 mg/kg/day revealed slightly but statistically higher neutrophil counts when compared with the Control. This difference did not show a dose response, so no effect of treatment was inferred.

All other differences were minor, confined to one sex or did not show a relationship to treatment and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Biochemical examination of blood plasma in Week 6 for males and on Day 13 of lactation for females did not reveal any findings that were considered related to treatment.

The investigations in Week 6 for males revealed slightly lower, but statistically significant low creatinine concentrations when compared with Control in males receiving 100, 330 or 1000 mg/kg/day, and statistically significantly higher phosphorus concentrations in males receiving 100 mg/kg/day.

The biochemical examination of the blood plasma on Day 13 of lactation for females revealed, when compared with the Controls, statistically significantly lower A/G ratios in females receiving 1000 mg/kg/day. This difference occurred in isolation, so no effect of treatment was inferred.

All other differences were minor, confined to one sex or did not show a relationship to treatment and were therefore attributed to normal biological variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The clinical condition of the animals, their behavior in the arena, sensory reactivity, grip strength and motor activity were all unaffected by treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The evaluation of organ weights of males after 5 weeks of treatment and of females on Day 13 of lactation revealed no differences when compared with the Controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item related effects were observed.

All macroscopic observations were generally consistent with the usual pattern of findings in animals of this strain and age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Change related to treatment with octadecan-l-ol, ethoxylated, phosphates was seen in the jejunum.

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of various cell types present within the different stages. No cell or stage specific abnormalities were noted. The qualitative examination of the ovary also revealed no abnormality.

Jejunum
Lymphangiectasis were seen in all males and some females given 1000 mg/kg/day.

All other microscopic findings were considered incidental and unrelated to test item.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in offspring on Day 13 of age. There was therefore no requirement to measure T4 in the samples obtained from offspring on Day 4 of age or from the adult females and none of the TSH (thyroid stimulating hormone) samples required analysis.

Details on results:

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in the Day 13 offspring. There was therefore no requirement to measure T4 in the samples obtained from offspring on Day 4 of age or from the adult females and none of the TSH (thyroid stimulating hormone) samples required analysis.

Number of abortions:

not examined

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the number of implantations, litter size or the growth of the offspring.

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

One female receiving 1000 mg/kg/day failed to litter and was found to be pregnant at macroscopic examination on Day 25 of gestation with two implantations and one dead pup in the right uterine horn.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

effects on pregnancy duration

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

number of abortions

organ weights and organ / body weight ratios

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The clinical condition of the offspring, offspring survival and sex ratio were unaffected by parental treatment.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The clinical condition of the offspring, offspring survival and sex ratio were unaffected by parental treatment.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Litter size and offspring body weight on Day 1 of age were unaffected by treatment; offspring weight gain was statistically significantly higher at 100, 330 and 1000 mg/kg/day. This is considered to be related to the slightly lower litter sizes in these groups and not an effect of treatment with the test item.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

Ooffspring survival was unaffected by parental treatment.

External malformations:

no effects observed

Description (incidence and severity):

Ano-genital distance of both male and female offspring on Day 1 of age and male nipple counts on Day 13 of age showed no effects of parental treatment.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The clinical condition of the offspring were unaffected by parental treatment.

Macroscopic examination of offspring that died prior to the scheduled termination or were killed on Day 13 of age did not reveal any findings that were considered related to parental treatment at any dose level.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

Key result

Developmental effects observed:

no

Conclusions:

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and also for reproductive/developmental toxicity was considered to be 1000 mg/kg/day.

Executive summary:

The purpose of this study was the assessment of systemic toxic potential in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item, octadecan-1-ol, ethoxylated, phosphates, by oral gavage administration for at least five weeks.

Three groups of ten male and ten female rats received octadecan-1-ol, ethoxylated, phosphates at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration. 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. The F1 generation 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, corn 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, estrous cycles, pre-coital interval, mating performance, fertility, gestation length,thyroid hormone analysis,organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age. Blood samples were collected from selected offspring on Day 4 (not analyzed) and Day 13 of age for thyroid hormone analysis. 

The clinical condition of the offspring, offspring survival and sex ratio were unaffected by parental treatment. Litter size and offspring body weight on Day 1 of age were unaffected by treatment; offspring weight gain was statistically significantly higher at 100, 330 and 1000 mg/kg/day. This is considered to be related to the slightly lower litter sizes in these groups and not an effect of treatment with the test item.

Ano-genital distance of both male and female offspring on Day 1 of age and male nipple counts on Day 13 of age showed no effects of parental treatment.

Macroscopic examination of offspring that died prior to the scheduled termination or were killed on Day 13 of age did not reveal any findings that were considered related to parental treatment at any dose level.

Reproductive performance, fertility and offspring survival were unaffected by parental treatment. There was no effect of treatment on the number of implantations, litter size or the growth of the offspring.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and also for reproductive/developmental toxicity was considered to be 1000 mg/kg/day.