Octadecanoic acid, reaction products with triethylenetetramine

Endpoint summary

Description of key information

A NOAEL for systemic toxicity of 1000 mg/kg bw/day was established for Octadecanoic Acid, reaction products with triethylenetetramine based on information from an OECD 422 guideline study.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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

Study period:

2017-08-09 to 2017-11-20

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:

29 July 2016

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Envigo Netherlands, Kreuzelweg 53, 5961 NM Horst, Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
7 to 8 weeks old
- Weight at study initiation:
200 to 225 g for males and 175 to 200 g for females
- Fasting period before study: no
- Housing: From arrival to mating, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm.
During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor.
After mating, the males were re-caged as they were before mating.
The femaleswere transferred to individual solid bottomed cages (measuring 42.5×26.6×18.5 cm) for the gestation period, birth and lactation.
- Diet (e.g. ad libitum):
ad libitum, A commercially available laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019, SettimoMilanese (MI), Italy)
- Water (e.g. ad libitum):
ad libitum to each cage via water bottles
- Acclimation period:
An acclimatisation period of approximately 3 weeks was allowed before the start of treatment,
during which time the health status of the animals was assessed by thorough observations.


ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22°C ± 2°C
- Humidity (%):
55% ± 15%,
- Air changes (per hr):
15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light):
artificial light for 12 hours

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered orally by gavage at a dose volume of 10mL/kg body weight.

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of Octadecanoic Acid, reaction products with triethylenetetramine was suspended in the vehicle. The formulations were prepared daily (concentrations of 10, 30 and 100 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Justification for use and choice of vehicle (if other than water): standard vehicle
- Concentration in vehicle: The formulations were prepared daily (concentrations of 10, 30 and 100 mg/mL) in 0.5% aqueous solution of carboxymethylcellulose
- Amount of vehicle (if gavage): 10 mL/ kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The proposed formulation procedure for the test item was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration and homogeneity) to confirm that the method was suitable.
Final results for all levels were within the acceptability limits for concentration (85-115%) and homogeneity (CV< 10%).

Duration of treatment / exposure:

Females of the main groups were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for a total of 42 to 64 days.
Females of the recovery groups were treated for a total of 56 days.
Males of the main groups were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 30/31 days.
Males of the recovery group were treated for a total of 28 days.

Frequency of treatment:

once a day, 7 days a week

Dose / conc.:

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

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:

Each main group comprised 10 male and 10 female rats (Groups 1 to 4).
Control and high dose level included 5 additional animals per sex to be sacrificed after 4 weeks of recovery (Groups 5 and 6).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels have been selectedbased on information from a preliminary study.
- Rationale for animal assignment:
The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights.
- Post-exposure recovery period in satellite groups: recovery from any treatment-related effects during a period of 4 weeks.

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Animals were checked twice daily for mortality. Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed weekly from allocation to termination. Females were weighed weekly
from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum.
Dams were also weighed on Days 1, 4, 7, 13 post partum and just before necropsy.
Recovery groups: Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

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: no feeding study
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: no feeding study

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: not applicable

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): not applicable

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrificial procedure
- Anaesthetic used for blood collection: Yes; under isofluorane anaesthesia
- Animals fasted: Yes; under condition of food deprivation
- How many animals:
5 males and 5 females randomly selected from each main group
At the end of the recovery period, blood samples were also taken from all surviving animals of the recovery groups
- Parameters checked in table [No.1] were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrificial procedure
- Anaesthetic used for blood collection: Yes; under isofluorane anaesthesia
- Animals fasted: Yes; under condition of food deprivation
- How many animals:
5 males and 5 females randomly selected from each main group
At the end of the recovery period, blood samples were also taken from all surviving animals of the recovery groups
- Parameters checked in table [No.1] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
-- Time schedule:
Once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination.
- Dose groups that were examined:
5/10 animals of the main groups selected randomly and in all animals of the recovery groups
Once duringWeek 4 of recovery, the MA was also performed in all recovery animals.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The clinical history of the adult animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
Changes were noted, the requisite organs weighed (Table 2)(excluding animals sacrificed for humane reasons or found dead) and the required tissue samples preserved in fixative and processed for histopathological examination.

HISTOPATHOLOGY: Yes
Samples of all the tissues listed table 2 were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).

Statistics:

Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of theWilliams test.
The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if ‘n’ was more than 5.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicological relevance were observed in males and females of main and recovery groups.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One low dose female was sacrificed for humane reasons on Day 23 post coitum for difficulty in parturition.
Pallor, decreased activity and piloerection were observed in this animal prior to sacrifice.
No treatment-related changes were observed after gross and histopathology evaluation.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No difference in body weights was recorded in animals of both sexes compared to the control group, throughout the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No effects on food consumption were observed in either males or females of main and recovery groups.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Slight lymphopenia was recorded in some females dosed at 300 and 1000mg/kg/day. However, due to the absence of dose-relation, these findings cannot be conclusively attributed to treatment.
Recovery phase (recovery phase animals): Lymphopenia and monocytopenia were still observed in females dosed at 1000mg/kg/day.
Coagulation: Dosing phase; no changes of toxicological relevance were observed.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Dosing phase
Triglycerides were decreased in a number of animals of all treated groups, with no doserelation.
In addition, glucose was increased in males receiving 1000mg/kg/day and in some females from all treated groups, with no dose-relation.
Recovery phase
Triglycerides showed complete reversibility in animals of both sexes. In males, glucose was comparable with controls, even though data were similar to those recorded at the end of treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Observation of animals at removal from the cage and in an open arena did not reveal changes attributable to the test item.
No treatment-related alterations in motor activity, grip strength and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in absolute and relative organ weight in all treatment groups of both sexes, when compared to the concurrent control data.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross pathology examination revealed no treatment-related changes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathology evaluation revealed no treatment-related changes. Inparticular, no treatmentrelated findings were noted in the thyroid gland of all males and females of all main groups, when compared to their controls.
In addition seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Histopathology evaluation revealed no treatment-related changes.

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone determination:
Parental animals
No changes of toxicological relevance were observed at the end of dosing and recovery phases in parental males and females from main and recovery phase groups.

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: no treatment related effects observed

Key result

Critical effects observed:

no

Conclusions:

Based on the results of the present study, the NOAEL for general toxicity was considered to be 1000 mg/kg/day for both males and females.

Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening study according to OECD guideline 422, Octadecanoic Acid, reaction products with triethylenetetramine was administered to 10 Sprague-Dawley rats /sex/ at dose levels of 0, 100, 300 and 1000 mg/kg bw/day by gavage.

Males of the main groups were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 30/31 days. Females of the main groups were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for a total of 42 to 64 days.

Additionally a recovery group was included (5 rats/sex) for control and high dose with a 4 week treatment-free period in order to assess any delayed toxicity or recovery from any adverse effects observed during the dosing phase. Males of the recovery group were treated for a total of 28 days and killed after 4 weeks of recovery period. Females of the recovery groups were treated for a total of 56 days. The females were sacrificed after 4 weeks of recovery period.

One low dose female was sacrificed for humane reasons on Day 23 post coitum for difficulty in parturition. Pallor, decreased activity and piloerection were observed in this animal prior to sacrifice. No treatment-related changes were observed after gross and histopathology evaluation. 

No clinical signs of toxicological relevance were observed in males and females of main and recovery groups.

No treatment-related alterations in motor activity, grip strength and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment.

No difference in body weights was recorded in animals of both sexes compared to the control group, throughout the study.

No effects on food consumption were observed in either males or females of main and recovery groups.

In main phase animals slight lymphopenia was recorded in some females dosed at 300 and 1000 mg/kg/day. However, due to the absence of dose-relation, these findings cannot be conclusively attributed to treatment. In recovery phase animals Lymphopenia and monocytopenia were still observed in females dosed at 1000 mg/kg/day. No changes of toxicological relevance were observed for coagulation parameters.

Triglycerides were decreased in a number of animals of all treated groups during dosing phase, with no dose relation. In addition, glucose was increased in males receiving 1000 mg/kg/day and in some females from all treated groups, with no dose-relation. Triglycerides showed complete reversibility in recovery animals of both sexes. In males of recovery group, glucose was comparable with controls, even though data were similar to those recorded at the end of treatment.

 

Terminal body weight of treated animals of the three main groups, as well as the high dose recovery group was comparable to the control group. No treatment-related changes were observed in absolute and relative organ weight in all treatment groups of both sexes, when compared to the concurrent control data.

 

Gross pathology examination revealed no treatment-related changes. Histopathology evaluation revealed no treatment-related changes. In particular, no treatment related findings were noted in the thyroid gland of all males and females of all main groups and of one male and one female pup of all dams on Day 14 post partum, when compared to their controls. In addition seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted.

 

Conclusion

No treatment-related effects indicating systemic toxicity were observed in male or female animals from main and recovery groups at any of the dose levels investigated (100, 300 and 1000 mg/kg/day).

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 1000 mg/kg/day for males and females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD 422 Guideline with GLP

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

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening study according to OECD guideline 422, Octadecanoic Acid, reaction products with triethylenetetramine was administered to 10 Sprague-Dawley rats /sex/ at dose levels of 0, 100, 300 and 1000 mg/kg bw/day by gavage.

Males of the main groups were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 30/31 days. Females of the main groups were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for a total of 42 to 64 days.

Additionally a recovery group was included (5 rats/sex) for control and high dose with a 4 week treatment-free period in order to assess any delayed toxicity or recovery from any adverse effects observed during the dosing phase. Males of the recovery group were treated for a total of 28 days and killed after 4 weeks of recovery period. Females of the recovery groups were treated for a total of 56 days. The females were sacrificed after 4 weeks of recovery period.

One low dose female was sacrificed for humane reasons on Day 23 post coitum for difficulty in parturition. Pallor, decreased activity and piloerection were observed in this animal prior to sacrifice. No treatment-related changes were observed after gross and histopathology evaluation. 

No clinical signs of toxicological relevance were observed in males and females of main and recovery groups.

No treatment-related alterations in motor activity, grip strength and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment.

No difference in body weights was recorded in animals of both sexes compared to the control group, throughout the study.

No effects on food consumption were observed in either males or females of main and recovery groups.

In main phase animals slight lymphopenia was recorded in some females dosed at 300 and 1000 mg/kg/day. However, due to the absence of dose-relation, these findings cannot be conclusively attributed to treatment. In recovery phase animals Lymphopenia and monocytopenia were still observed in females dosed at 1000 mg/kg/day. No changes of toxicological relevance were observed for coagulation parameters.

Triglycerides were decreased in a number of animals of all treated groups during dosing phase, with no dose relation. In addition, glucose was increased in males receiving 1000 mg/kg/day and in some females from all treated groups, with no dose-relation. Triglycerides showed complete reversibility in recovery animals of both sexes. In males of recovery group, glucose was comparable with controls, even though data were similar to those recorded at the end of treatment.

 

Terminal body weight of treated animals of the three main groups, as well as the high dose recovery group was comparable to the control group. No treatment-related changes were observed in absolute and relative organ weight in all treatment groups of both sexes, when compared to the concurrent control data.

 

Gross pathology examination revealed no treatment-related changes. Histopathology evaluation revealed no treatment-related changes. In particular, no treatment related findings were noted in the thyroid gland of all males and females of all main groups and of one male and one female pup of all dams on Day 14 post partum, when compared to their controls. In addition seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted.

 

Conclusion

No treatment-related effects indicating systemic toxicity were observed in male or female animals from main and recovery groups at any of the dose levels investigated (100, 300 and 1000 mg/kg/day).

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 1000 mg/kg/day for males and females.

Justification for classification or non-classification

Repeated dose toxicity of Octadecanoic Acid, reaction products with triethylenetetramine was assessed based on data from a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening study.

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg/day, the highest tested dose.

Evaluating information from the sub-acute toxicity study a classification with STOT-RE according to GHS Regulation EC No 1272/2008 for Octadecanoic Acid, reaction products with triethylenetetramine is not warrant.