Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Preliminary results from EOGRTS 2, 2’-(Octadec-9-enylimino) bisethanol shows no efefcts on reproduction parameters, whereas the symptoms of general toxicity (mainly BW and local effects stomach) are the same in F0 and F1 animals.

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
other: experimental study - Interim
Remarks:
Study is not completed; Final report is scheduled for April 2021 - See attchement. Reported are available preliminary results.
Adequacy of study:
key study
Study period:
25 March 2020 - December 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
Adopted 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
As requested by ECHA decision TPE-D-2114449858-29-01/F of 21 November 2018
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Strain RccHan™;WIST; Supplier Envigo (UK) Limited
Age ordered 22-28 Days; Body weight range 30 g per sex; Males unrelated to females
Sex:
male/female
Details on test animals or test system and environmental conditions:
Age at start of treatment (F0) : Approximately 4-5 weeks of age (approximately 14-15 weeks of age at pairing)
Husbandry conditions
Animal facility Limited access - to minimize entry of external biological and chemical agents.
Air supply Filtered, not recirculated.
Temperature Maintained within the range of 20-24ºC.
Relative humidity Maintained within the range of 40-70%.
Monitored daily. Excursions outside these ranges documented in the study data.
Lighting 12 hours light : 12 hours dark.
Alarm systems Activated on ventilation failure and when temperature/humidity limits exceeded.
Electricity supply Public supply with automatic stand-by generators.
Cage material: Polycarbonate
Cage flooring: Solid polycarbonate
Grid bottomed cages will be suspended above absorbent paper which will be changed daily during pairing.
Bedding : Bedding type Softwood based bark-free fiber, sterilized by autoclaving.
Diet supply : Diet name SDS VRF1 Certified, pelleted diet.
Availability Non-restricted.
Certification Before delivery each batch of diet is analyzed by the supplier for various nutritional components and chemical and microbiological contaminants.
Supply Potable water from the public supply.
Availability Non-restricted via polycarbonate bottles with sipper tubes.
Certification Certificates of analysis are routinely received from the supplier.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Daily oral gavage; 4 mL/kg/day.
Individual dose volume Calculated from the most recently recorded scheduled body weight.
Formulation:
A daily record of the usage of formulation will be maintained based on weights before and after dosing. The difference between actual and expected usage will be monitored as a check of correct administration.
Formulations are stirred using a magnetic stirrer before and throughout the dosing procedure.
Vehicle: Arachis oil
Frequency of preparation Weekly
Storage of formulation Refrigerated at 2 to 8°C.
[stability has been demonstrated for 24 hours at ambient temperature (15 to 25C)]

Dose (mg/kg/day): 0, 30, 70, 150.
Concentrations:
Group 1 Vehicle
Group 2 2,2'-(Octadec-9-enylimino)bisethanol; 7.5 mg/mL
Group 3 2,2'-(Octadec-9-enylimino)bisethanol; 17.5 mg/mL
Group 4 2,2'-(Octadec-9-enylimino)bisethanol; 37.5 mg/mL
Details on mating procedure:
F0 pairing commences After 10 weeks of treatment.
Male/female ratio 1:1 (sibling pairing will not be permitted).
Duration of pairing Up to 2 weeks.
Daily checks for evidence of mating
Day 0 of gestation When positive evidence of mating detected.
Male/female separation Day when mating evidence detected.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
To be reported.
Duration of treatment / exposure:
F0 animals For 10 weeks before pairing until termination after litters are weaned.
F1 animals From weaning until termination of respective cohort.
Frequency of treatment:
Daily
Details on study schedule:
Litters culled to 8 (where possible 4 males and 4 females) on Day 4 of age.
Formal commencement of the F1 generation is on a nominal Day 28 of age (where possible 28±2 days of age for selected F1 animals).
2 males and 2 females from each litter; Microchipped day 18-21 and separated from littermates on day 21.
Termination:
F0 Females: 28 day post partum (wk: 10 + 3 + 2 = 15 plus some days mating: ca. 100 days);
F0 Males: After weaning of the F1 animals.
Unselected offspring On Day 4 and Day 22 of age
F1A adult animals At approximately 13 weeks of age
F1B adult animals At approximately 14 weeks of age

Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
LD
Dose / conc.:
70 mg/kg bw/day (nominal)
Remarks:
MD
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
HD
No. of animals per sex per dose:
F0: 25
1A: 20
1B: 20
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry:
- Other:
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week
Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation for F0 (and F1 females if mating triggered).

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 Males: Each week & Before necropsy.
F0 Females: Each week until mating; Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating.
Days 1, 4, 7, 14, 21 and 28 post partum.
Before necropsy
F1: From nominal 4 weeks of age, twice during Week 1 of the F1 generation and weekly thereafter.

FOOD CONSUMPTION: yes
- Food consumption calculated as g food/kg body weight/day: Yes, per cage.

WATER CONSUMPTION : No
Oestrous cyclicity (parental animals):
Dry smears For 15 days before pairing, using cotton swabs.
Wet smears After pairing until evidence of mating confirmed, using pipette lavage. For four days before scheduled termination (nominally Days 25 to 28 post partum).
Sperm parameters (parental animals):
Yes: (F0 and F1A)
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Litters culled to 8 (where possible 4 males and 4 females) on Day 4 of age.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
- F0 Females: 28 day post partum (wk: 10 + 3 + 2 = 15 plus some days mating: ca. 100 days);
- F0 Males: After weaning of the F1 animals.
- Unselected offspring On Day 4 and Day 22 of age
- F1A adult animals At approximately 13 weeks of age
- F1B adult animals At approximately 14 weeks of age

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were weighed and prepared for microscopic examination, respectively:
Adrenals
Brain (cerebellum, cerebrum, midbrain)
Epididymides
Heart (including auricular and ventricular regions)
Kidneys
Liver (section from two lobes)
Ovaries with oviduct
Pituitary
Prostate– dorsolateral and ventral combined
Seminal vesicles (with coagulating gland)
Spleen
Testes
Thymus
Thyroid with parathyroids
Uterus with cervix
Prepared for microscopic examination, not weighted
Abnormalities
Cecum
Colon
Duodenum
Esophagus
Eyes
Femurs - (longitudinal section through joint)
Ileum
Jejunum
Lungs (section from two major lobes including bronchi)
Optic nerves
Pancreas
Rectum
Sciatic nerves
Skeletal muscle
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Sternum - bone marrow
Stomach
Trachea
Urinary bladder
Vagina
Vas Deferens
Postmortem examinations (offspring):
Unselected F1 offspring (Day 22 of age)
10 male and 10 females per group; one male or one female from each litter to ensure that all litters are represented.
The tissues indicated below were weighed and prepared for microscopic examination, respectively:
The tissues indicated below were weighed and fixed, respectively:
Brain (cerebellum, cerebrum, midbrain)
Spleen
Thymus
Only fixed, not weighted
Abnormalities
Epididymides
Ovaries
Pituitary
Prostate
Seminal vesicles
Skin with mammary glands (inguinal area)
Testes
Uterus with cervix and oviducts
Vagina

F1A Adult animals (Cohort 1A)
The tissues indicated below were weighed and prepared for microscopic examination, respectively:
Adrenals
Brain (cerebellum, cerebrum, midbrain)
Epididymides
Heart (including auricular and ventricular regions)
Kidneys
Liver (section from two lobes)
Lymph nodes – mesenteric & left axillary
Ovaries with oviduct
Pituitary
Prostate– dorsolateral and ventral combined
Seminal vesicles (with coagulating gland)
Spleen
Testes
Thymus
Thyroid with parathyroids
Uterus with cervix
Prepared for microscopic examination, not weighted
Abnormalities
Cecum
Colon
Duodenum
Esophagus
Eyes
Femurs - (longitudinal section through joint)
Ileum
Jejunum
Lungs (section from two major lobes including bronchi)
Optic nerves
Pancreas
Rectum
Sciatic nerves
Skeletal muscle
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Sternum - bone marrow
Stomach
Trachea
Urinary bladder
Vagina
Vas Deferens

Statistics:
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.
Alternative or additional methods may be carried out if deemed appropriate following data review.
Reproductive indices:
Percentage mating
Conception rate
Fertility index
Gestation length
Gestation index
Offspring viability indices:
Post implantation survival index
Live birth index
Viability index
Lactation index
Sex ratio
Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs observed at either routine physical examination or in association with dose administration that were considered to relate to administration of the test item.
A few animals receiving 150 mg/kg/day were observed with increased salivation in association with dose administration; this sign is often seen in association with oral gavage administration and is attributed to the taste of the formulation rather than an effect of treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
A total of four animals (one male and three females) were euthanized for welfare reasons.
· One male from Group 1 (animal no. 18, that received 30 mg/kg/day) was dispatched for poor clinical condition and was sacrificed in an unscheduled non-moribund on Day 43 of treatment.
· Three females (animal nos. 279, 280 and 278 that received 150 mg/kg/day) were sacrificed in an unscheduled non-moribund condition on Day 2 of lactation, 21 of gestation and 23 of gestation, respectively. The cause of death was attributed to poor clinical condition (animal nos. 18, 279 and 280) or due to urogenital lesions (consisted of moderate hemorrhage within the uterus that likely contributed to its moribund condition) (animal no. 278). However, no histopathological findings which could account for the poor clinical condition were observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the 10 week pre-pairing treatment period males receiving Oleyl-PFAEO had significantly low body weight gain and low mean absolute body weights; a dose response was apparent.
Body weight gain for females during Week 4 of treatment was significantly low at all dose levels; however the overall gain for females before paining showed no adverse effects of treatment.
After mating body weight was slightly low during Days 14-16 for females receiving 150 mg/kg/day; however for females that have reached Day 20 after mating the overall weight gain currently shows no adverse effect of treatment.
On Day 1 of lactation mean body weight for females receiving 150 mg/kg/day was low when compared with Controls (p<0.01), however overall the bodyweight gain at 70 or 150 mg/kg/day from Day 1-21 of lactation was high when compared with Control values (p<0.01); these differences were attributed to high weight gain during the first two weeks of lactation.

Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males receiving 70 or 150 mg/kg/day showed low food consumption from Week 1 of treatment (p<0.05/0.01); a dose response was apparent. Males at 30 mg/kg/day also showed occasions of low consumption during Week 2 (p<0.05), Week 5(p<0.01) and Week 9 (p<0.05) of treatment.
Before pairing females at 150 mg/kg/day showed low food consumption during Weeks 4, 5, 7, 8 and 9 (p<0.05/0.01) and overall consumption was approximately 86 % of Controls; food consumption for females receiving 30 or 70 mg/kg/day was unaffected by treatment.
After mating females receiving 150 g/kg/day showed slightly low food consumption, with statistical significance achieved on Days 2-5 and 12-19 (p<0.05/0.01); food consumption at 30 or 70 mg/kg/day was similar to Controls.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males and females receiving 70 or 150 mg/kg/day and males receiving 30 mg/kg/day slightly but statistically significantly shorter prothrombin clotting times when compared with Controls.
At 150 mg/kg/day the mean neutrophil count for males and females was high when compared with Controls, the difference attaining statistical significance for the male animals (p<0.01).
Females at 150 mg/kg/day also showed a high mean eosinophil count when compared with Controls (p<0.05).
Males at 150 mg/kg/day also showed:
· low mean haemoglobin (p<0.01)
· low mean cell haemoglobin (p<0.01)
· low mean cell volume (p<0.05)
· high platelet count (p<0.01)
Contrary to male animals, females at all dose levels showed high mean cell haemoglobin (p<0.05) and a longer activated partial thromboplastin time (p<0.01) when compared with Controls.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Alanine transaminase activity for males and females receiving 150 mg/kg/day was elevated when compared with Controls (p<0.05). Males at 150 mg/kg/day also showed low total protein and albumin (p<0.01), with both males and females showing a high A/G ratio at 70 mg/kg/day (p<0.05) and 150 mg/kg/day (p<0.01).
Other statistically significant differences were as follows:
· gamma-glutamyl transpeptidase activity at 150 mg/kg/day increased for male animals only (p<0.01);
· bile acid concentration at 150 mg/kg/day was high for males (p<0.01) but low for females (p<0.05);
· urea concentration at 70 and 150 mg/kg/day were high for males only;
· cholesterol levels at all dose levels were slightly low for females only (p<0.05);
· sodium concentrations at 150mg/kg/day were high for males (p<0.05) and low for females (p<0.05).
Urinalysis findings:
not specified
Description (incidence and severity):
No results yet
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Preliminary.
In unscheduled and terminal sacrifice animals, 2,2’-(Octadec-9-enylimino) bisethanol-related microscopic findings were observed in the stomach (both sexes administered 30, 70 or 1570 mg/kg/day), jejunum (both sexes administered 150 mg/kg/day and males administered 70 mg/kg/day) and duodenum (males administered 150 mg/kg/day).
The predominant microscopic finding in the stomach was characterized as diffuse minimal to moderate epithelial hyperplasia of the non-glandular region associated with erosion/vesicle or ulceration. Edema and mixed inflammation were considered secondary to the erosion/vesicle or ulceration. This finding correlated with the 2,2’-(Octadec-9-enylimino) bisethanol-related macroscopic observation of edema and thickness of the non-glandular mucosa.
Foamy cells accumulation observed within the jejunum (both sexes) and duodenum (males only) correlated with the 2,2’-(Octadec-9-enylimino) bisethanol-related macroscopic observation of abnormal color and thickness.
All other microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected in the Han Wistar rat.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Not yet examined
Reproductive performance:
no effects observed
Key result
Dose descriptor:
LOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
gross pathology
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Not yet completed:
Clin.signs: None
BW: lower at MD (♂ -7.1%) & HD (♀-5.7% ; ♂ -16.0%);
Food consumption: lower in HD
AGD: No effects
Sex.maturation: Increased 3 days at HD
Haematology: shorter prothrombin dose response
Neutrophils/Leucocytes ♂↑, only marginal (N do not go above L)
Changes Hb profile: some stat.significance and dose response, but effects biologically limited. Within historical control?
Blood chemistry: ♂ ALP & ALAT↑
Urine: prot.↑
Organ weights:
Rel. kidney↑
Spleen ↑
Pituitary: ↑
Thymus ♂↓
Macropathology: increased incidence of macroscopic changes in the stomach at 70 or 150 mg/kg/day.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
gross pathology
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
70 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
70 mg/kg bw/day (nominal)
Treatment related:
yes
Conclusions:
No conclusions can be made on the basis of the available preliminary information. Howevere, the available data indicates that there are no effects observed on reproduction, whereas general toxicity is the same for F0 and F1.
Executive summary:

Preliminary results:

Results: Parental EOL

Mortality:Dispatched following “General poor clinical condition”:

Grp

Animal

Day

Cause

Comments

1

M#18

day 43

 

Terminal signs included gasping, rales and partially closed eyelids. Macroscopic findings at necropsy were unremarkable and limited to enlarged mandibular lymph nodes.

4

F#280

GD 20

 

In addition to the deterioration in general condition this female lost 4g in body weight and showed reduced food consumption from GD18-20. 18 implantations; 17 grossly normal fetuses and one resorbing implant – fetal weights were not recorded.

 

F#278

GD 23

 

Urogenital lesions (consisted of moderate hemorrhage within the uterus that likely contributed to its moribund condition)

 

F#279

LD 2

 

 

Fertility:

Seven females (animal no. 220 that received Arachis oil, animal no. 235 at 30 mg/kg/day, animal nos. 251, 252 and 256 at 70 mg/kg/day and animal nos. 292 and 305 at 150 mg/kg/day) failed to litter and with the exception of female no 256 which had two implantation scars, no scars were evident for the other females and as such they were considered to have failed to conceive (not pregnant); there were no histopathological findings, which could account for the suspect fertility for these female animals.

Not pregnant

Contr:

F#220

M#19:bilateral marked tubular atrophy/degeneration of the testes with sperm absence within epididymides

LD:

F#235

To be examined

MD:

F#251, #252, #256 (two implantation scars)

To be examined

HD:

F#292no histopathological abnormalities

F#305no histopathological abnormalities

M#91:no histopathological abnormalities
M#100: no histopathological abnormalities

 

Macroscopic Observations

At the terminal sacrifice, thickeness was noted for the stomach of some animals administered 30, 70 or 150 mg/kg/day. This finding was associated in females with edema (few animals across all treated groups) and correlated with the 2,2’-(Octadec-9-enylimino) bisethanol-related microscopic observation of diffuse hyperplasia of the nonglandular mucosa, erosion/vesicles, ulceration, edema and mixed inflammation.

 

The jejunum presented abnormal color in some males and females administered 150 mg/kg/day and two males administered 70 mg/kg/day. This finding was associated with thickeness in some animals administered 150 mg/kg/day and correlated with the 2,2’-(Octadec-9-enylimino) bisethanol-related microscopic observation of accumulation of foamy cells.

 

All other macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for Han Wistar rats of this age; therefore, they were considered not 2,2’-(Octadec-9-enylimino) bisethanol related.

Microscopic Observations

In unscheduled and terminal sacrifice animals, 2,2’-(Octadec-9-enylimino) bisethanol-related microscopic findings were observed in the stomach (both sexes administered 30, 70 or 1570 mg/kg/day), jejunum (both sexes administered 150 mg/kg/day and males administered 70 mg/kg/day) and duodenum (males administered 150 mg/kg/day).

 

The predominant microscopic finding in the stomach was characterized as diffuse minimal to moderate epithelial hyperplasia of the non-glandular region associated with erosion/vesicle or ulceration. Edema and mixed inflammation were considered secondary to the erosion/vesicle or ulceration. This finding correlated with the 2,2’-(Octadec-9-enylimino) bisethanol-related macroscopic observation of edema and thickness of the non-glandular mucosa.

 

Foamy cells accumulation observed within the jejunum (both sexes) and duodenum (males only) correlated with the 2,2’-(Octadec-9-enylimino) bisethanol-related macroscopic observation of abnormal color and thickness.

 

All other microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected in the Han Wistar rat.

 

CONCLUSION

Following oral gavage administration of 2,2’-(Octadec-9-enylimino) bisethanol to Han Wistar rats, treatment related changes were observed in the stomach (consisting of epithelial hyperplasia of the non-glandular region associated with erosion/vesicle or ulceration, edema and mixed inflammation), jejunum and duodenum (consisting of foamy cells accumulation within the lamina propria).

 

In view of the findings, it is recommended that histopathological examination is extended to the duodenum of male animals and the stomach and jejunum of both sexes from the low and intermediate dose groups.

 

Results: Maturation F1

Mortality:

(nM - nF)

C

30

70

150

F1 - A1

0 -1

0 - 0

0 -2(1)

1- 0

F1 - B1

0 - 0

1- 0

0 - 0

3(2)- 0

LD♂ 1B 512, d47 (welfare - General poor clinical condition)

HD♂ 1B 550, d25 (welfare - trauma tibia, not test substance related)

HD♂ 1B 559, d10 (found dead - perforated esophagus - not test substance related)

C♀ 1A 616, d29 (dispatched for suspect fertility: failed to get vaginal opening - vulva was imperforate)

MD♀ 1A 655, d11 (welfare perforated esophagus - not test substance related)

 

Clin.signs:None

BW:lower at MD (♂ -7.1%) & HD (♀-5.7% ; ♂ -16.0%);

Food consumption:lower in HD

AGD:No effects

Sex.maturation:Increased 3 days at HD

 

Haematology:shorter prothrombin dose response

Neutrophils/Leucocytes ♂↑, only marginal (N do not go above L)

Changes Hb profile: some stat.significance and dose response, but effects biologically limited. Within historical control?

Blood chemistry:♂ ALP & ALAT↑

Urine:prot.↑

 

Organ weights:

Rel. kidney↑

Spleen ↑

Pituitary: ↑

Thymus ♂↓

 

Macropathology:increased incidence of macroscopic changes in the stomach at 70 or 150 mg/kg/day.

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
125 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
High quality study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The OECD408 90 day dosing study in rats on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 showed a lack of any adverse effects on the reproductive organs. There were no effects on the weight of the reproductive organs or seen when examined histopathologically. Also in addition the females were examined for any abnormality in the oestrus cycle and the males for abnormality in the spermatogenic cycle. No abnormalities were seen which supports the lack of potential for reproductive toxicity in the adult rats.

 

The in-life phase of an EOGRTS study with 2, 2’-(Octadec-9-enylimino) bisethanol is just completed, and the results are being collected and evaluated. Interim results from the available data indicates that there are no effects observed on reproduction, whereas general toxicity is the same for F0 and F1.

 

Justification for selection of Effect on fertility via inhalation route:

The low vapour pressure of the substance means inhalation is not considered to be relevant route of exposure so not testing is required.

 

Justification for selection of Effect on fertility via dermal route:

The corrosive properties of this substance mean the repeated dose dermal studies are not scientifically justified due to concerns for animal welfare.

Effects on developmental toxicity

Description of key information

There is an OECD414 pre-natal developmental toxicity study on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9. This study showed no indication of developmental toxicity in the foetuses, the NOEL being 150mg/kg.  There was however also no indication of toxicity in the pregnant females.  This study does not include dosing during the implantation period, the only available information concerning this is read across to the OECD422 study on Ethanol, 2,2’-iminobis-,N-coco alkyl derivs CAS No 61791-31-9.  This substance is predominantly C12 but does contain >10% C18 while 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9  is predominantly C18.  The available study is an OECD 422 reproductive screening test.  There were some indications of foetotoxicity (increased post implantation loss)  in this study but this was considered not sufficient for classification, it has been proposed to do a reproduction study to establish if this is also seen with 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
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:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test”
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology Studies, 12 NohSan No 8147 (24 November 2000)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 190g to 269g.

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

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

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

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

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

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

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

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

Instrumental Setip

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


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

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

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

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

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

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

Implantation types were divided into:

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

Late Death: Separate embryonic/fetal and placental tissue visible

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

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


Left Horn Cervix Right Horn

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

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

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

Probability values (p) are presented as follows:

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

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

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

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

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

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

Introduction

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

 

The study was designed to comply with the following guidelines:

 

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

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

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

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

  

Methods….

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

Clinical signs, body weight change, food and water consumptions were monitored during the study. 

 

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

 

Results….

Adult Responses

Mortality

There were no unscheduled deaths during the study.

 

 Clinical Observations

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

  

Body Weight

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

 

 Food Consumption

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

 

 Water Consumption

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

 

 Post Mortem Studies

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

 

 Litter Responses

Litter Data and Litter Placental and Fetal Weights

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

 

 Fetal Examination

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

 

 Conclusion

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

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no 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 and replaces the read across to Ethanol, 2,2’-iminobis-,N-coco alkyl derivs CAS No 61791-31-9 for developmental toxicity but it does not cover the period immediately after implantation so it does not supersede the read across to the OECD422 study where increased post implantation loss was seen. This will be resolved when the proposed reproduction study on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 is available.
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

The OECD414 pre-natal development study included dose levels of 15, 50 and 150mg/kg bodyweight of 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9, 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 embryofoetal 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.

 

Conclusion

 

The No Observed Effect Level (NOEL) for the pregnant females and the survival, growth and embryofoetal development of the offspring was considered to be 150 mg/kg bw/day.2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9, did not show any indications of potential for developmental toxicity in this study. The proposed reproduction study will allow an assessment if the increased post implantation loss seen with the read across substance 2,2’-iminobis-,N-coco alkyl derivs CAS No 61791-31-9 is also seen with this substance.

 

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

We have a Klimisch 1 full GLP compliant OECD414 study on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9.

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

The low vapour pressure of the substance means inhalation is not considered to be relevant route of exposure so not testing is required.

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

The corrosive properties of this substance mean the repeated dose dermal studies are not scientifically justified due to concerns for animal welfare.

Justification for classification or non-classification

No effects on reproduction have been observed in an EOGRTS on 2, 2’-(Octadec-9-enylimino) bisethanol.

 

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. Until the reproduction study is available it is not possible to know if the increased post implantation loss see in the OECD422 study with the read across substance Ethanol, 2,2’-iminobis-,N-coco alkyl derivs CAS No 61791-31-9 will also be seen with this substance. However the evidence from the OECD414 study indicates that 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 does not induce developmental toxicity in rats, therefore it is clear that it is not necessary to classify this substance for reproductive or developmental toxicity based on the current data. A final conclusion concerning the post implantation loss and its relevance to this substance will be possible when the reproduction study is completed.

Additional information