Amides, tall-oil fatty, N,N'-(iminodi-2,1-ethanediyl)bis-, phosphates

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

Type of information:

experimental study

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

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:

2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes]
- Age at study initiation: approx 10 weeks at start of treatment
- Weight at study initiation: males 413-495 g, females 224-287 g
- Fasting period before study: no
- Housing: up to 3 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or indivdually housed
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY:
animals received ssniff SM R/M complete diet for rats and mice, Soest, Germany
animals received tap water from municipal supply.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 - 24.9
- Humidity (%): 31 - 64
- Air changes (per hr): 15 -20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: propylene glycol containing 1% polysorbat 80

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:


The test item was formulated in the vehicle propylene glycol containing 1% polysorbat 80 (as a visibly stable homogenous suspension). Formulations were prepared daily at appropriate concentrations in the vehicle.

The calculated volume of Propylene glycol / 1% Tween 80 vehicle was added into a beaker containing the calculated amount of test item, it was mixed vigorously for at least an hour by a magnetic stirrer. Then formulation was incubated overnight at room temperature and mixed again by a magnetic stirrer for approximately 2 hours. Formulations were mixed continually with a magnetic stirrer until the end of the treatment procedures

- Concentration in vehicle: 20, 60, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg body weight
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test item formulations for concentration and homogeneity was performed photometrically using a validated NIR method. Recovery of nominal concentrations ranged from 99% to 107%.

Duration of treatment / exposure:

males were dosed for 28 days starting 14 days prior to mating. Females were dosed for 14 days prior to mating, during mating, gestation until day 13 post partum.

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

according to guideline OECD 422 (2016)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: food consumption was determined

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: just before necropsy
- Anaesthetic used for blood collection: Yes pentobarbital
- Animals fasted: Yes
- How many animals: 5 per sex and dose group
- Parameters checked in table [No.?] were examined: see Table on haematology attached as background material

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: just before necropsy
- Animals fasted: Yes
- How many animals: 5 per sex and dose group
- Parameters checked in table [No.?] were examined.: see Table on clinical chemistry and urinalysis attached as background material

URINALYSIS: Yes
- Time schedule for collection of urine: just before necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked in table [No.?] were examined: see Table on clinical chemistry and urinalysis attached as background material

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity.

IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table)

see "pathology and histopathology investigations" attached as background material

Statistics:

The statistical evaluation of data (labelled as † in the lists below) was performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS v9.2 (when using Provantis).
In case of the SPSS PC+4.0 program package, the heterogeneity of variance between groups was checked by Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, then Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of non-normal distribution, the non-parametric method of Kruskal-Wallis One- Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. The Chi-squared test was used for non-continuous data.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

one animal of the low dose group died due to a gavage accident.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

see Tables and Figures on body weight attached as background material

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

see Tables and figures on food consumption attached as background material

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly lower (p<0.05) prothrombin time (PTT) was recorded in the Low dose males. As the recorded value is within the historical control range and there was no dose response, this difference was considered to not reflect any adverse effects of the test item.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no statistically different results in any of the dose groups compared to the control.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly higher urine volume was collected from the Low dose males (p<0.05) and from the High dose females (p<0.01). The collected urine volume is usually highly variable. All study data was considered to be normal, therefore this numerical difference was considered to not reflect any adverse effects of the test item. This fact is confirmed by the lack of any supporting evidence of any changes in these animals (clinical chemistry, other urinalysis parameters, etc.)

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly higher (p<0.01) absolute brain weights in the male Mid dose group and significantly lower (p<0.05) relative kidney weights in the female Low and High dose groups were recorded. However, as the observed values were near the middle of the historical control range and there were no clear dose responses observed in these parameters, these statistical differences were considered to have no toxicological significance.

see Tables on weights of organs attached as background material


Besides this, there were no statistically different results in any other of the dose groups compared to the control.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item-related findings were seen at dose levels up to 1000 mg/kg bw/day.

Based on the low incidence and/or severity and/or distribution cross control and dosed animals the following observations were considered incidental or a common background: minimal or slight tubular basophilia in the kidney (#1001, 4506), minimal multifocal casts in the kidney (#1511), minimal tubular multifocal mineralization in the kidney cortex (#1504, 4503), minimal focal congestion or haemorrhage in the stomach (#1506), minimal multifocal congestion or haemorrhage in the thymus (#1001, 1504).

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL = highest dose tested

Key result

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

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 was derived at the highest dose of 1000 mg/kg/day for systemic toxicity in adult male and female rats after four to six weeks of oral exposure to WS400103. This does not necessitate any classification regarding repeated exposure according to European classification rules [Regulation (EC) 1272/2008].