Fatty acids, C14-18 and C16-18-unsatd., 2-phenoxyethyl esters, maleated

Endpoint summary

Description of key information

Combined Repeated Dose Oral (Gavage) Toxicity Study with Reproduction/Developmental Toxicity Screening in Rats to OECD 422:
Doses / Study Design:                                               0 (vehicle control), 100, 300, 1000 mg/kg bw/day, with 5 toxicity subgroup animals/sex/dose.
Treatment period, toxicity subgroup animals:      Daily, for five consecutive weeks
Sacrifice, toxicity subgroup animals:                      Week 6
Treatment with WS400109 was generally well tolerated at each dose level with no toxicologically significant systemic effects.
For general toxicity, the NOAEL was based on the following differences from concurrent or historical controls:
- Overall bodyweight gain over 5 treatment weeks higher in females at 1000 mg/kg/day
- Motor activity scores higher for females at 1000 mg/kg/day
- WBC, lymphocyte and basophil counts higher in males at 300 and 1000 mg/kg/day
- Slightly higher incidence of macroscopically thickened duodenum and jejunum with corresponding histopathology (epithelial hyperplasia) at 1000 mg/kg/day
- Slightly higher incidence of epithelial hyperplasia and hyperkeratosis in the non-glandular stomach at 1000 mg/kg/day.
Conclusion: NOAEL = 300 mg/kg/day for subacute (5 weeks) repeated dose toxicity testing.

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 screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study fully adequate for assessment. The study was conducted according to an internationally accepted technical guideline and in compliance with GLP in a recognized contract research organization.

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)

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. 71 days.
- Weight at treatment start: Males: minimum 327 g, maximum 411 g,
Females: minimum 230 g, maximum 271 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 polycarbonate 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: 6 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.
* Due to clotting of some of the initial blood samples, the bleed for heamatology was repeated without overnight deprivation of food.

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 these 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 bodyweight. 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 during the 7-day range-finding study:
Endpoint study record "7.5.1 Repeated dose toxicity: oral - 7d_range-finding_gavage_HLS_GAH0134".
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).
- Mean concentrations (verified for first and last treatment week) of the test material formulations were confirmed at each dose level.
Chemical analysis confirmed that the mean concentrations of WS400109 in prepared formulations were 94% to 104% of the corresponding
nominal concentration, thus confirming acceptable accuracy of formulation for dosing of the animals.
- Homogeneity and stability of test material formulations at 2 and 200 mg/L and at storage and handling conditions similar to those adopted for dosing of the animals were confirmed.

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): 42 to 46 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)

Remarks:

Doses / Concentrations:
0 (vehicle control), 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

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 (F0) 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 (gavage) toxicity study in the rat in which dose levels of 100, 300 or 1000 mg/kg/day did not have any overt treatment-related effects on young adult animals (females nulliparous and non-pregnant).

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: About weekly throughout the study.
Body weight, Toxicity Females: About weekly throughout the study.
Body weight, Repro. Females: Weekly for pre-pairing period; on gestation days 0, 6, 13, 20; on lactation days 1, 4 & 7.
- Food consumption, all males: Weekly for pre-pairing period and for the period after mating.
Food cons., Toxicity Females: About 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.

Haematological 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, triglycerides, total bilirubin, bile acids, sodium, potassium, chloride,
calcium, inorganic phosphorus, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase.

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 (F0) and toxicity subgr. females: Killed in Week 6, after completion of the Treatment Week 5 investigations.
- reproductive subgr. females & offspring: Killed on Day 7 post partum.
(1 control & 1 low dose female were killed on the respective Day 25 after mating, as they had failed to litter)
(1 mid & 1 high dose dam and their litter were killed on the respective Lactation Day 3 for animal welfare
reasons. Their offspring had poor survival prognosis.)

Gross pathology:
- adult/parental animals: Full macroscopic examination.
- offspring, Lactation Day 7: Careful external macroscopic examination of all survivors for gross abnormalities.
- externally abnormal offspring
and premature deaths: Internal 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:
- all males (F0) +
toxicity subgroup females: Adrenals, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles with coagulation gland,
spleen, testes, thymus, uterus with cervix & oviducts.
- dams: Ovaries

Histopathology:
- toxicity subgroups*: The following organs were microscopically observed for the control and 1000 mg/kg bw/day groups:
Brain, eyes, pituitary gland, thyroid with parathyroids, heart, thymus, liver, spleen, adrenals, kidneys, testes,
epididymides, ovaries, lung, trachea, oesophagus, 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, sternum with marrow, seminal vesicle & coagulation gland, prostate.
In addition, any gross lesions for all adult animals from all dose groups were examined by light microscopy.

* Histopathology examination only on control & high dose groups, 5 males and 5 females per group

- reproductive subgroups Any 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, litter size, survival indices & clinical pathology data:

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

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

Grip strength, motor activity, survival indices and clinical pathology data
if 75% of the data (across all groups) were the same value, pairwise comparison of each dose group against 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 a Wald chi-square test

For statistical references, see next field.

Clinical signs:

no effects observed

Description (incidence and severity):

attributable to treatment with the test material

Mortality:

no mortality observed

Description (incidence):

attributable to treatment with the test material

Body weight and weight changes:

no effects observed

Description (incidence and severity):

attributable to treatment with the test material

Food consumption and compound intake (if feeding study):

no effects observed

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):

, but not toxicologically significant

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

, but not toxicologically significant

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

motor activity increased in high dose females

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no deaths or clinical signs attributable to treatment with WS400109.

NEUROBEHAVIOUR
Effects on sensory reactivity or grip strength attributable to treatment with the test material were not evident. During treatment week 5, motor activity tended to be higher in females treated at 1000 mg/kg/day than in concurrent controls, whereby group mean values of the 1000 mg/kg/day dose group often were higher than the upper limit of the historical reference range.

BODYWEIGHT, WEIGHT GAIN AND FOOD CONSUMPTION
Food consumption was unaffected by treatment with WS400109. In treated males and main phase females bodyweight, in general, was unaffected. In nulliparous, nonpregnant females (toxicology subgroup) overall bodyweight gain over 5 treatment weeks was higher at 1000 mg/kg/day than in concurrent controls (x 1.39) but this was considered to represent normal biological variation.

CLINICAL PATHOLOGY
Toxicologically significant changes in haematology or clinical chemistry (blood plasma) parameters were not evident. After 5 weeks of treatment at 300 and 1000 mg/kg/day, total leukocyte (WBC), lymphocyte and basophil counts statistically significantly higher than in concurrent controls in males and bilirubin and cholesterol statistically significantly lower in females were not considered to represent toxicologically significant effects. In addition, potassium statistically significantly higher than in concurrent controls in both sexes at 1000 mg/kg/day were not considered to represent adverse effects. All of these changes were well within the corresponding historical reference range.

ORGAN WEIGHTS
Organ weights were unaffected by treatment.

GROSS PATHOLOGY AND HISTOPATHOLOGY (NON-NEOPLASTIC)
Thickened duodenum and jejunum were macroscopically apparent in two males and one female receiving 1000 mg/kg/day. Histopathological correlates to these findings were epithelial hyperplasia in the duodenum and jejunum in both males and in the duodenum in this female. In addition, epithelial hyperplasia and hyperkeratosis were seen at a slightly higher incidence in the nonglandular stomach in both sexes at 1000 mg/kg/day than in concurrent controls.

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

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

Justification for classification or non-classification

In the OECD 422 Study, the NOAEL for general toxicity was 300 mg/kg/day for male and female rats with no toxicologically significant systemic effects up to the highest dose level of 1000 mg/kg bw/day. The NOAEL does not necessitate any classification regarding repeated exposure according to European classification rules [DIRECTIVE 67/548/EEC and REGULATION (EC) 1272/2008], because WS400109 did not induce any adverse effects at dose levels corresponding to the limits recommended by the European Regulations for classification.