Fatty acids, tall-oil, compds. with oleylamine

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental toxicity study

Type of information:

experimental study

Remarks:

The experimental study was performed with a structural analogue; read-across from results of this study is justified (see below).

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Sprague Dawley rats, strain: Crl:CD(SD) with appropriate range of bodyweight at study start.
- Source: Charles River (UK) Ltd.
- Age at treatment start: Ca. 70 days.
- Weight at treatment start: Males: minimum 341 g, maximum 390 g,
Females: minimum 212 g, maximum 277 g.
- Housing Inside a barriered rodent facility:
all animals pre-pairing + toxicity subgroups: In groups up to 5 by sex in solid floor polycarbonate cages.
during pairing (1 male+1 female/cage): In RB3 modified polypropylene cages with stainless steel grid-floor over absorbent paper-lined trays.
males after pairing: In groups up to 5 in solid floor polycarbonate cages.
females during gestation and lactation: Females housed individually (+litter) in solid floor polycarbonate cages.
- Bedding material (in solid floor cages): Wood based bedding, sterilised by autoclaving before use.
- Cage enrichment: Aspen chew block + plastic shelter (except during pairing or post gestation day 20).
- Diet (ad libitum): Standard rodent diet (SDS VRF1 Certified) without antibiotic, chemotherapeutic or prophylactic agent.
- Fasting (diet withheld): Main phase males and Toxicity phase females overnight before blood sampling for clinical pathology.
- Water (ad libitum): Potable drinking water from the public supply.
- Acclimation period: 7 days before treatment start, under laboratory conditions.

Routine analysis of the batch of diet used and water, chew blocks and bedding material did not provide evidence of contamination that might have prejudiced the study.

IN-LIFE DATES:
- Duration of test, males & toxicity phase females: Five weeks
Duration of test, main phase females (i.e. reproductive subgroup): From 14 days prior to pairing to day 7 of lactation.
Duration of test, offspring: From birth to day 7 of lactation.

ENVIRONMENTAL CONDITIONS

Air conditioned room kept at positve pressure without re-circulation of the filtered fresh air supplied to the room.
Controlled environment, environmental conditions were set at:
- Temperature (°C): 21 ± 2°C
- Relative Humidity (%): 40 to 70%
- Photoperiod (artificial lighting): 12 hrs day / 12 hrs night
- Rate of air exchange: At least 15 changes/h
Deviations from the target ranges for temperature and relative humidity were not evident.

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

- Concentration in vehicle: The concentration of the test material in vehicle varied between dose groups thus allowing constant dosage volume in terms of mL/kg bw/day.
- Amount (dose volume by gavage): 5 mL/kg bw/day.
Actual dose volumes were calculated at about weekly or shorter intervals accounting for the latest body weight. Litter animals were not dosed.

- For concentrations of test material in vehicle at different dose levels, see Table 1 in "Any other information on materials and methods incl. tables"

- Justification for choice of vehicle:
The suitability of propylene glycol as a vehicle was established visually and by chemical analysis during the 7-day range-finding study:
Endpoint study record "7.5.1 Repeated dose toxicity: oral - 7d_range-finding_gavage_HLS_GAH0029".
In addition, in the present main study, concentrations of dose formulations were chemically analysed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Chemical analysis of test material formulations by high performance liquid chromatography coupled with a mass spectrometer (HPLC-MS/MS).
- Concentrations (verified at first, second and last treatment week) of the test material formulations were lower than expected from nominal concentrations (see Table 1).
- Mean concentrations of WS400151 in prepared formulations attained by chemical analysis were 70.5% to 87.2% of the corresponding nominal concentration. Evaluation of a number of aspects of the analytical and formulation procedures did not lead to identification of the cause for the lower concentrations achieved by chemical analysis. In order to take a conservative approach, it was concluded that the concentrations determined by chemical analysis reflected the concentrations of test substance formulation administered to the animals.

Duration of treatment / exposure:

- Treatment period, males & toxicity phase females: Daily, for five consecutive weeks, in males commencing 14 days prior to pairing
- Treatment period, main phase females (i.e. reproductive subgroup): 44 to 47 days (from 14 days prior to pairing to day 6 of lactation)
- Offspring were not dosed

Frequency of treatment:

Daily, 7 days/week (during parturition, dosing omitted as appropriate)

Dose / conc.:

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

Remarks:

dose levels derived from chemical determination of administered dose formulations

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

Toxicity phase animals: */ 5 females
Main phase animals (i.e. reproductive subgroups): 10 males / 10 females
*Explanatory note by the notifier:
Examinations assigned to the toxicity phase females to meet the requirements of a 28-day repeat dose oral toxicity study were also assigned to 5 (for some examinations to 10) main phase males per dose group. Therefore, these 5 main phase males per dose group are called also "toxicity subgroup" in the present robust study summary for clarification. After pairing with main phase females, all males were killed at the same time (Week 6).

Control animals:

yes, concurrent vehicle

Details on study design:

This study was conducted to examine both repeated dose toxicity and  reproductive/developmental toxicity as an OECD screening combined study
(OECD 422 test guideline).  Therefore, animals initially entering the study were divided into toxicity subgroup animals (toxicity phase) and reproductive subgroup animals (main phase), whereby 5 of the 10 F0 males (used for pairing) per dose group formed the toxicity male subgroups.

Dose selection was based on the results of a 7-day preliminary oral toxicity study in young adult rats (females nulliparous and non-pregnant) in which a NOAEL could not be established at dose levels of 100, 300 or 1000 mg/kg/day.

Positive control:

Not included in the study.

Observations and examinations performed and frequency:

Clinical observations performed and frequency:
- Clinical signs : At least twice a day (before and after administration)
- Detailed physical examination
and arena observations: Before treatment start and at least once a treatment week.
- Functional Observation Battery:* During treatment week 5 (before dosing) on all toxicity subgroup animals (5 males + 5 females/group).
- Body weight, all males: Weekly throughout the study.
Body weight, Toxicity Females: Weekly throughout the study.
Body weight, Repro. Females: Weekly for pre-pairing period; on gestation days 0, 6, 13, 20; on lactation days 1 & 7.
- Food consumption, all males: Weekly for pre-pairing period and for the period after mating.
Food cons., Toxicity Females: Weekly throughout the study.
Food cons., Repro. Females: Weekly for pre-pairing period, during gestation for days 0-6, 6-13, 13-20, during lactation for days 1-4 & 4-7.

* FOB including sensory reactivity tests (approach, touch, auditory startle reflex, tail pinching), grip strength and motor activity.

Hematological examinations (only for toxicity subgroup animals) during treatment week 5 after functional observation battery:
Red blood cell count, reticulocyte count, white blood cell count, platelet count, hemoglobin concentration, hematocrit value, differential leukocyte counts,
protrombin time, activated partial thromboplastin time, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration.

Blood (plasma) chemical examinations (only for toxicity subgroup animals) during treatment week 5 after functional observation battery:
Total protein, albumin, A/G ratio, urea, creatinine, glucose, total cholesterol, total bilirubin, bile acids, sodium, potassium, chloride,
calcium, inorganic phosphorus, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase.

This study was conducted to examine both repeated dose toxicity and  reproductive/developmental toxicity as an OECD screening combined study
(OECD 422 test guideline).  Therefore, some of the examinations were confined to toxicity subgroup animals, as indicated above.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, see below
WEIGHING OF ORGANS: Yes, see below
HISTOPATHOLOGY: Yes, see below

Terminal sacrifice
- all males and toxicity subgr. females: Killed in Week 6, after completion of the Treatment Week 5 investigations.
(2 high dose females died from intubation error on treatment day 32)
- reproductive subgr. females & offspring: Killed on Day 7 post partum.
(1 high dose female died from intubation error on treatment day 14)
(1 low and 1 high dose females without viable litter were killed on Day 25 after evidence of mating)

Gross pathology:
- adult/parental animals: Full macroscopic examination with tissue collection.
- offspring: Full macroscopic examination including assessment of the presence of milk in the stomach, where possible.
(Missing or grossly autolysed or cannibalised offspring could not be examined).

Organs Weights:
- main phase and tox. subgr. adults: Adrenals, brain, epididymides, heart, kidneys, liver, lungs & bronchi, ovaries, pituitary, prostate, seminal vesicles
& coagulation gland, spleen, testes, thymus, thyroid with parathyroids, uterus with cervix & oviducts.

Histopathology:
- toxicity subgroups: The following organs were microscopically observed for the control and 75 mg/kg bw/day groups:
Brain, eyes, Harderian glands, optic nerves, pituitary gland, thyroid with parathyroids, heart, thymus, liver, spleen,
adrenals, kidneys, testes, epididymides, ovaries, lung, trachea, esophagus, stomach, duodenum, jejunum, ileum,
caecum, rectum, colon, Peyer's patch, lymph node (axillary, mesenteric), urinary bladder, uterus (with cervix &
oviducts), vagina, spinal cord, sciatic nerve, skeletal muscle, skin with mammary glands, sternum with marrow,
seminal vesicle & coagulation gland, prostate. In addition, the jejunum, mesenteric lymph nodes and duodenum
were also examined for the 7.1 and 21.9 mg/kg bw/day toxicity subgroups and any gross lesions for all adult
animals.
- reproductive subgroups: Gross lesions from all adult animals from all dose groups were examined by light microscopy.

Other examinations:

Reproductive and developmental toxicity parameters (addressed in separate endpoints).

Statistics:

Statistical analysis of grip strength, motor activity, bodyweight, food consumption, organ weight, oestrous cycles, mating performance, gestation length, fertility, litter size, survival indices & clinical pathology data:

- Parametric analysis, if Bartlett's test for variance homogeneity not significant at the 1% level.
F1 approximate test for monotonicity of dose-response. If this F1 test not significant at the 1% level, then Williams' test for a monotonic trend.
If this F1 test significant, suggesting that the dose-response was not monotone , then Dunnett's test instead.

- Non-parametric analysis, if Bartlett's test still significant at the 1% level following logarithmic and square-root transformations.
H1 approximate test for monotonicity of dose-response. If this H1 test not significant at the 1% level, then Shirley's test for a monotonic trend.
If this H1 test significant, i.e. dose-response not monotone, then Steel's test instead.

Grip strength, motor activity, survival indices & clinical pathology data
If 75% of the data (across all groups) were the same value, pairwise comparison of each dose group with the control by Fisher’s Exact tests.

Organ weight data
Covariance analysis using terminal bodyweight as covariate (Angervall & Carlstrom, 1963)

Sex ratio
Analysis by generalised mixed linear model with binomial errors, a logit link function and litter as a random effect (Lipsitz et al 1991).
Comparison of each treated group with control using Wald chi-square test

Gestation length
Exact (or asymptotic) 2-tailed Linear-by-linear test (Cytel 1995), with equally spaced scores*

Oestrous cycles
Exact (or asymptotic) 1-tailed (upper tail) Linear-by-linear test (Cytel 1995)*

Numbers mating, conceiving & fertile
Exact 1-tailed (lower tail) Cochran-Armitage test (Cytel 1995)*

* If significant (p<0.05), exclusion of the highest dose group and re-testing until no longer significant (p≥0.05).

References, next field.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

attributable to treatment with the test material

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

only at 75 mg/kg/day

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

only at 75 mg/kg/day; during weeks 1 & 2 in both sexes and during gestation and lactation

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):

only at 75 mg/kg/day; counts of monocytes slightly high, of total white cells and neutrophils marginally high

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

only at 75 mg/kg/day; in jejunum, mesenteric lymph node and duodenum

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

at 21.9 and 75 mg/kg/day; in jejunum, mesenteric lymph node and/or duodenum

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

CLINICAL SIGNS, NEUROBEHAVIOUR AND MORTALITY
Three high dose females (2 of the toxicity subgroup and 1 of the reproductive subgroup) were found dead following intubation errors. One low and one high dose females without viable litter were killed on Day 25 after evidence of mating. Mortality or clinical signs attributable to treatment with the test material or effects on sensory reactivity, grip strength or motor activity were not evident.

BODY WEIGHT AND WEIGHT GAIN
At 75 mg/kg/day, overall bodyweight gain was low in males during the 5-week treatment period and in females during the first two treatment weeks and during gestation.

FOOD CONSUMPTION
At 75 mg/kg/day, food consumption was slightly low in both sexes during the first two treatment weeks, and in dams it was lower than concurrent controls, overall during gestation and lactation. At lower dose levels (7.1 and 21.9 mg/kg/day) foodconsumption was unaffected by treatment with the test material.

CLINICAL PATHOLOGY
At 75 mg/kg/day, monocyte counts were slightly higher than concurrent controls in both sexes and total white cell and neutrophil counts were marginally higher than concurrent controls. Toxicologically significant effects on clinical chemistry parameters in blood plasma were not evident.

GROSS PATHOLOGY
After five treatment weeks at 75 mg/kg/day, the jejunum of all surviving animals was thickened, the mesenteric lymph nodes were enlarged in 2 males and 2 females and were pale in in 3 males, and the duodenum was thickened in 1 male and 2 females. At lower dose levels toxicologically relevant macroscopic lesions were not evident.

Dark areas seen in the mesenteric lymph nodes in 2 males at 75 mg/kg/day were probably due to sinus erythrocytosis and erythrophagocytosis which is a common background finding and are unlikely to be related to treatment with WS400151. In addition, necropsy revealed that the oesophagus of the three high dose decedent animals was perforated thus confirming that dosing errors caused their premature death.

ORGAN WEIGHTS
There were no adverse effects on organ weight.

HISTOPATHOLOGY: NON-NEOPLASTIC
The presence of foamy macrophages in the mucosa of the jejunum in males at 21.9 mg/kg/day and both sexes at 75 mg/kg/day and in the duodenum in two females at 75 mg/kg/day, and foamy histiocytosis in the sinuses of the mesenteric lymph nodes in both sexes at 21.9 and 75 mg/kg/day (see Table 2) were attributed to treatment with the test material and were considered potentially adverse, because the aetiology of the foamy macrophages in the gut mucosa was uncertain.

OTHER RESULTS
Reproductive and developmental toxicity parameters are addressed in separate endpoints.

Key result

Dose descriptor:

NOAEL

Effect level:

> 7.1 - < 21.9 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

duodenum

jejunum

mesenteric lymph node

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

 

Table 2: Summary of Treatment Related Histopathology Findings for Animals Dying during the Study or Killed after at least 5 Weeks of Treatment
Group/sex	1/M	2/M	3/M	4/M	1/F	2/F	3/F	4/F
Dose (mg/kg/day)	0	7.1	21.9	75	0	7.1	21.9	75
Jejunum
Foamy macrophages, mucosa
Minimal Slight	0 0	0 0	2 0	5 4	0 0	0 0	0 0	5 1
Total	0	0	2	9	0	0	0	6
Number of tissues examined	5	5	5	10*	5	5	5	6*
Mesenteric Lymph Node
Foamy sinus histiocytes
Minimal Slight	0 0	0 0	4 0	5 3	0 0	0 0	4 0	2 3
Total	0	0	4	8	0	0	4	5
Number of tissues examined	5	5	5	9*	5	5	5	5*

 

* Including the two decedent females (94 and 95) from Week 5 of treatment and some animals where the tissues were examined

because abnormalities were reported at macroscopic examination.

 

In addition, foamy macrophages, minimal in degree, in the mucosa of the duodenum were seen in two high dose females (75 mg/kg/day), but not in any other treated or control animals.

 

Conclusions:

Foamy macrophages in the mucosa of jejunum and/or duodenum and foamy histiocytosis in sinuses of mesenteric lymph nodes at 21.9 and 75 mg/kg/day associated with high monocyte count, low bodyweight gain and low food consumption at 75 mg/kg/day were considered potentially adverse, because the aetiology of the foamy macrophages in the gut mucosa was uncertain. The histopathology findings may represent accumulation of WS400151 or of a metabolite in the macrophages or phospholipidosis. Based on these findings the NOAEL for WS400151 after 5 treatment weeks was the low dose level of 7.1 mg/kg/day .

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

7.1 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

well performed guideline conform oral repeat dose study (performed with read across substance)

System:

gastrointestinal tract

Organ:

duodenum

jejunum

mesenteric lymph node

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The substance WS400101 is the salt (physical mixture) of fatty acids, tall oil, and octadec-9-enylamine. This chemical composition is similar to the substance WS400151 (CAS 147900-93-4) which is the salt of fatty acids, C18-unsaturated, trimers and octadec-9-enylamine.

Based on the result of the oral repeat dose study performed with WS400151 it was concluded that the toxic effects observed in this study were due to octadec-9-enylamine; fatty acids, C18-unsaturated, trimers do have a low toxicological potential. Because WS400101 also is a fatty acids salt of octadec-9-enylamine it is expected that the same toxic effects would be observed in an oral repeat dose study due to octadec-9-enylamine; the fatty acids do have a low toxicological potential. The two substances WS400101 and WS400151 both contain approx. the same amount octadec-9 -enylamine.

 

Based on these reasons read-across from the subacute oral toxicity study performed with WS400151 was made for WS400101. Further read-across with regard to toxicity after repeated oral dosing is made from the harmonised classification and labelling that was adopted for octadec-9-enylamine based on many toxicological data available on this substance and similar long-chain alkylamines (Regulation (EC) 1272/2008). These long-chain alkylamines are classified for specific target organ toxicity - repeated exposure STOT RE 2. This classification also is applied to WS400101.

Justification for classification or non-classification

WS400101 has to be classified as STOT RE 2.