Isooctadecanoic acid, reaction products with tetraethylenepentamine

Endpoint summary

Description of key information

Several repeat dose toxicity studies are available for the registered substance. No adverse effect was observed at any dose level tested. The NOAEL is 1000 mg/L (the highest dose tested).

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

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3/01/2006-2/16-2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to an internationally approved protocol and followed Good Laboratory Practice standards.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Animals in the 12 and 50 mg/kg dose group were underdosed. This did not affect the outcome of the study since the highest dose level was properly administered and no adverse effects were observed. Other deviations did not affect the outcome either.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Age at study initiation: 8 weeks
- Weight at study initiation: 204-270 g (male); 151-207 (female)
- Housing: individually in wire mesh suspension cages
- Diet (e.g. ad libitum): ad libitum except prior to blood collection
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Test article formulations were prepared weekly as single formulations for each dosage level through Apr 7. For the remainder of the study, the 12 adn 50 mg/mL formulations were prepared every other day and the 200 mg/mL formulation continued to be prepared weekly. Formulations were divided into aliquots for daily dispensation and stored at room temperature with continuous stirring.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples (1mL) for concentration analysis were collected from each test group during the first week and last week. By extrapolation from the available information, Group 2 animals were under-dosed by up to 80% during the first two weeks and by up to 22.2% during the last two weeks. Group 3 animals were under-dosed by up to 50% during the first two weeks. The 1000 mg/kg/day group was dosed within acceptable ranges for the entire 28 days of dosing.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 60, 250, 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

14/sex/dose in control and high dose; 7/sex/dose in the low and mid dose

Control animals:

yes, concurrent vehicle

Details on study design:

Following 4 weeks of dose administration, 7 rats/sex/group were euthanized; the remaining 7 rats/sex in the control and high dose groups were euthanized following a 14-day nondosing (recovery) period.

Observations and examinations performed and frequency:

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded weekly. Functional observational battery and locomotor activity data were recorded for 7 animals/sex/group prior to the initiation of dose administration and during study week 3. Clinical pathology evaluations (hematology, serum chemistry and urinalysis) were performed for all rats assigned to the primary (study week 4) and recovery (study week 6) necropsies.

Sacrifice and pathology:

Complete necropsies were conducted on all animals, and selected organs were weighed. Selected tissues were examined microscopically from all animals in the control and 1000 mg/kg/day groups. Gross lesions were examined from all animals in the 60 and 250 mg/kg/day groups.

Statistics:

All statistical tests were performed using appropriate computing devices or programs.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

There was a low incidence of chronic and chronic active inflammation in the lungs that were considered due to accidental aspiration of dosing formulation and not a manifestation of systemic toxicity.

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 signs of toxicity were observed in any of the parameters evaluated at any dose level tested.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No signs of toxicity were observed in any of the parameters evaluated at any dose level tested.

Critical effects observed:

not specified

Conclusions:

Under the conditions of this study, EC 701-204-9 did not exhibit adverse toxic effects and the NOAEL >1000 mg/kg/day (the highest dose tested)

Executive summary:

The registered substance was administered orally (gavage) to Crl:CD(SD) rats for 28 consecutive days at dose levels of 0, 60, 250, and 1000 mg/kg/day. All animals survived until scheduled necropsy. There were no test article related clinical findings or effects on body weight, food consumption, functional observational battery evaluations, locomotor activity, clinical pathology parameters or organ weights at any dose level. There were no test article related macroscopic or microscopic changes observed either. The No Observed Adverse Effect Level is greater than 1000 mg/kg/day.

Reference 2

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Qualifier:

according to guideline

Guideline:

other: Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.26: “Sub-chronic Oral Toxicity Test: Repeated dose 90- day toxicity study in rodents". Official Journal of the European Union No. L142, Ma

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Strain: Crl: WI(Han)
Condition: Outbred, SPF-Quality
Source: Charles River Deutschland, Sulzfeld, Germany or Charles River
Laboratories France, L'Arbresle Cedex, France.

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were allowed to acclimate to the Test Facility toxicology accommodation for 12 days before the commencement of dosing.

The conditions for animal room environment were as follows:
Temperature: Targeted: 18 to 24°C
Actual mean: 21 to 24°C
Humidity: Targeted: 40 to 70%
Actual mean: 45 to 72%
Light Cycle: 12 hours light and 12 hours dark (except during designated procedures)
Ventilation: Ten or more air changes per hour
The values that were outside the targeted humidity range occurred for 4 out of 132 days with a maximum of 72% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study.

Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany
Type: Pellets
Frequency: Ad libitum, except during designated procedures.
Analysis: Results of analysis for nutritional components and environmental contaminants were provided by the supplier and are on file at the Test Facility. There were no known contaminants in the feed that would interfere with the objectives of the study.

Type: Municipal tap water.
Frequency/Ration: Freely available to each animal via water bottles.
Analysis: Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. There were no known contaminants in the water that could interfere with the outcome of the study.

For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted by study procedures/activities.

Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff once during the Dosing Period, due to a temporary change in the environmental conditions. The veterinary examination was documented in the study records.

Route of administration:

oral: gavage

Details on route of administration:

Dose Route: Oral gavage
Frequency: Once daily
Duration: 7 days a week for a minimum of 13 weeks.
Method: The first day of dosing was designated as Day 1. The dose formulations were stirred continuously during dosing. The doses were given using a plastic feeding tube.
Dose pot identification via Provantis was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

The following animals had reflux when dosed on the following days and therefore the actual dose volume was unable to be confirmed:
• Day 32 - Female No. 93 (1000 mg/kg/day)
• Day 57 - Female No. 97 (1000 mg/kg/day)

Vehicle:

corn oil

Remarks:

specific gravity 0.92

Details on oral exposure:

Dose Route: Oral gavage
Frequency: Once daily
Duration: 7 days a week for a minimum of 13 weeks.
Method: The first day of dosing will be designated as Day 1 (exception: Alternate animals used for replacement after Day 1 will assume the day of the animal being replaced). The dose formulations will be stirred continuously during dosing. The doses will be given using a plastic feeding tube.
Dose pot identification via Provantis will be used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Analytical verification of doses or concentrations:

yes

Remarks:

The objective of the analytical study was to determine the accuracy of the preparation and homogeneity of EC 701-204-9 in formulations. Analysis was based on the analytical method validated for the test item in vehicle in Test Facility Reference No. 2024

Details on analytical verification of doses or concentrations:

Instrument Acquity UPLC system (Waters, Milford, MA, USA)
Detector Xevo TQ-S mass spectrometer (Waters)
Column Acquity UPLC Protein BEH C4, 50 mm × 2.1 mm i.d., dp =1.7 μm (Waters)
Column temperature 40°C ± 1°C
Injection volume 1 μL
Mobile phase 0.1% formic acid in methanol
Flow 0.2 mL/min
MS detection
Ionization source ESI+
Cone voltage 40 V
Acquisition (SIR) m/z 366.2, m/z 486.5, m/z 730.9, m/z 971.3 and m/z 1237.6
Quantitation (SIR) m/z 730.9

Duration of treatment / exposure:

7 days a week for a minimum of 13 weeks.

Frequency of treatment:

Once 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:

50

10 males and 10 females for each dose.

For the 1000mg/kg/day dose, a "recovery group" with 5 males and 5 females.

Overall, 50 males and 50 females were used in this study.

Control animals:

yes, concurrent vehicle

Details on study design:

The objective of this study was to determine the potential toxicity of EC 701-204-9, when given orally by gavage for 90 days to Wistar Han rats and to evaluate the potential reversibility of any findings. In addition, a No Observed Adverse Effect Level (NOAEL) was evaluated.

Positive control:

No positive control was included.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily; from Day 1 at 0 to 1 hours post dose.
Animals will be observed within their cage unless necessary for identification or confirmation of possible findings. For observations that cannot be attributed to an individual animal due to social housing (e.g., watery feces), the observation will be recorded to each animal in the socialized group.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly; from at least Day 1 and throughout the study. In order to monitor the health status, animals may be weighed more often. Body weights will be kept in the study records.

FOOD CONSUMPTION : yes
Weekly; from at least Day 1 and throughout the study (Quantitatively measured per cage.)

WATER CONSUMPTION : Yes
- Time schedule for examinations: Regular basis throughout the study. (Water consumption was monitored by visual inspection of the water bottles.)

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretreatment Period - All Main Study and Recovery animals once (including spare animals). Dosing Period - All Group 1 and 4 Main Study animals during Week 13. The eyes were examined using an ophthalmoscope after application of a mydriatic agent (Tropicol, tropicamide 5 mg/mL solution, THEA Pharma, Wetteren, Belgium.
- Dose groups that were examined: All main study and recovery animals inclusing spare animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment and end of recovery. Sampled between 7.00 and 10.30 from the retro-orbital sinus under anesthesia using isoflurane.
- Anaesthetic used for blood collection: Yes (isoflurane (Abbott B.V., Hoofddorp, The Netherlands).
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: all main and recovery animals
- Parameters checked in table [5] were examined. Table 5 can be found on page 22, poinr 4.7.1.2 : hematology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment and end of recovery. Sampled between 7.00 and 10.30 from the retro-orbital sinus under anesthesia using isoflurane.
- Anaesthetic used for blood collection: Yes (isoflurane (Abbott B.V., Hoofddorp, The Netherlands).
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: all main and recovery animals
- Parameters checked in table [7] were examined. Table 7 can be found on page 23, poinr 4.7.1.4 : clinical chemistry.


PLASMA/SERUM HORMONES/LIPIDS: No

URIN ALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during the Dosing Period; the first 5 animals per sex per group during Weeks 12-13. These tests were performed after clinical observations.
- Dose groups that were examined: 5 animals per sex per dose group.
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
• hearing ability, pupillary reflex and static righting reflex (score 0 = normal/present, score 1 = abnormal/absent).
• fore- and hind-limb grip strength will be recorded as the mean of three measurements.
• locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations will be reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.

OTHER:

Salivation
Breathing, Abnormal Sounds

Fur, Loss
Fur, Thin Cover,
Skin, Lesion,
Skin, Scab,

Absolute Organ Weights
pididymisa
Gland, adrenala
Gland, Pituitary
Gland, prostate
Gland, seminal vesiclea
Gland, thyroid
Heart
Kidneya
Liver
Ovarya
Spleen
Testisa
Thymus
Uterus/cervix

Sacrifice and pathology:

Main Study and Recovery animals surviving until scheduled euthanasia were weighed, and euthanized using isoflurane, followed by exsanguination. Animals were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy.

Necropsy procedure : Necropsy, Organ weights, Tissue Collection, Histology processing, Microscopic evaluation.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.

Numerical data collected on scheduled occasions were analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. Values may also be expressed as a percentage of predose or control values when deemed appropriate. Inferential statistics were performed according to the matrix below when possible, but exclude semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical signs were noted for males dosed at 100 and 300 mg/kg/day and females dosed at 300 mg/kg/day.
Abnormal breathing sounds were noted in 1/15 males (No. 39) at 1000 mg/kg/day between Days 58-70. Based on its isolated incidence and the fact that this type of breathing abnormalities occasionally can be seen in oral gavage studies, it was considered to be of no toxicological relevance.

Salivation was noted in 2/15 males (Day 45) and 2/15 females (Days 30-33 or 89) at 1000 mg/kg/day, and in 3/10 females at 100 mg/kg/day (Day 26 or 30), next to 1/15 control females (Day 89). This finding of salivation was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and relatively minor severity of the effect, its time of occurrence (i.e. after dosing) and the fact that both control and treated animals were affected similarly.

Other findings (i.e. scabs and lesions (skin), and loss and thin cover of the fur) noted during the Dosing or Recovery Period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gain were considered to have been unaffected by the treatment with the test item.
Any statistically significant changes in mean body weight and body weight gain during the Recovery Period were considered to be unrelated to the test item since no test-item related changes were apparent during the Dosing Period, and changes were caused by slight body weight loss in control males (Days 113-119) or relative high body weight of the control females at the start of the Recovery Period

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption during the Dosing Period was comparable between all groups.
A relatively high food consumption was measured for one of the two cages for males dosed at
300 mg/kg/day between Days 71-78. Since males in the parallel cage from the same dose
group had a normal food consumption, and food consumption during the other weeks was
comparable to control animals, this was likely to be a spillage of food.
Food consumption during the Recovery Period was noted to be lower in females at
1000 mg/kg/day compared to concurrent controls. This was likely to be caused by the lower
body weight in this group compared to the concurrent controls, already present at the start of
the Recovery Period. Body weight gain during the Recovery Period was comparable or
slightly higher for Group 4 females compared to the concurrent control group, and food
consumption was stable during the Recovery Period and comparable to food consumption
during the Dosing Period. Therefore, no toxicological relevance was attached to this finding.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test item-related effects on functional observation parameters were noted at the end of the
90-days Dosing Period.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined
animals, and no effect on grip strength or motor activity was noted in males and females up to
1000 mg/kg/day.
No toxicological significance was attached to the observed increased mean total number of
ambulations during the motor activity measurement in females at 300 mg/kg/day, as this
change occurred in absence of a dose-related trend. Moreover, all groups showed a similar
motor activity habituation profile with a decreasing trend in activity over the duration of the
test period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted in hematological parameters.
The decreased mean basophil count at the end of the Dosing Period in all males dosed with
the test item was regarded to be the result of a slightly higher mean value of the control
animals.
Decreased means eosinophil (EOS) count in Recovery females at 1000 mg/kg/day was seen at
the end of the Recovery Period only and therefore considered to be not toxicological relevant.
Differences in hematology parameters, regardless of statistical significance, were considered
not test item-related based on the absence of a dose response, general overlap of individual
values with the range of control values, and/or were of a magnitude of change commonly
observed in rats under similar study conditions.

The apparent shorter mean prothrombin (PT) and activated partial thromboplastin times
(APTT) observed at the end of the Dosing Period in males at 100 and 300 mg/kg/day and
males at 300 and 1000 mg/kg/day, respectively, were unrelated to treatment with the test item
as no dose-response could be established. It was regarded to be the result of slightly high
control mean.
Female No. 56 at 0 mg/kg/day was noted to have a relative high APTT value, which was
considered to be not test item-related as this is a control animal.
The values achieving a statistical difference were considered not test item-related based on
the absence of a dose response, general overlap of individual values with the range of control
values, and/or were of a magnitude of change commonly observed in rats under similar study
conditions.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted in clinical biochemistry parameters at the end
of the Dosing Period
Any statistically significant changes in clinical biochemistry parameters at the end of the
Dosing Period were considered to be unrelated to treatment as these occurred in the absence
of a dose-related trend.
At the end of the Recovery Period, total bilirubin concentration was decreased in males
(0.74x of control) and creatinine was decreased in females (0.77x of control) of the
1000 mg/kg/day group. For these changes, or any other changes at the end of the Recovery
Period that did not reach statistical significance, a relationship with the test item was regarded
unlikely based on the lack of a test item-related effect in these parameters at the end of the
Dosing Period and in absence of corroborative findings at tissue level. Therefore, these results
were considered to be unrelated to treatment with the test item.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test item-related effects on functional observation parameters were noted at the end of the
90-days Dosing Period.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined
animals, and no effect on grip strength or motor activity was noted in males and females up to
1000 mg/kg/day.
No toxicological significance was attached to the observed increased mean total number of
ambulations during the motor activity measurement in females at 300 mg/kg/day, as this
change occurred in absence of a dose-related trend. Moreover, all groups showed a similar
motor activity habituation profile with a decreasing trend in activity over the duration of the
test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related alterations in organ weights.
All organ weight differences observed at the end of the 90-days treatment period in Main
animals, including those that reached statistical significance (Main females at 300 and 1000
mg/kg/day; lower pituitary gland weight, absolute and relative to body weight), were
considered not to be test item-related due to the lack of dose-related pattern, lack of
microscopic correlate and/or general overlap and variability in individual values.
After the 28-day dosing-free period, the 1000 mg/kg/day Recovery group males showed
statistically significant higher adrenal gland and thyroid gland weights (absolute and relative
to body weight). In the 1000 mg/kg/day group Recovery group females, there was a
statistically significant higher organ weight of the thyroid/parathyroid gland (relative to body
weight) and statistically significant lower organ weights of the heart (absolute) and liver
(absolute and relative to body weight). A relationship with the test-item was regarded unlikely
based on the lack of statistically significant changes and lack of test item-related microscopic
changes in these organs in Main group rats. Therefore, the differences in organ weights of
Recovery group animals in the high dose group compared to concurrent controls were
attributed to lower final body weights (females) and/or general overlap and variability in
individual values.

There were no test item-related alterations in organ weights.
All organ weight differences observed at the end of the 90-days treatment period in Main
animals, including those that reached statistical significance (Main females at 300 and 1000
mg/kg/day; lower pituitary gland weight, absolute and relative to body weight), were
considered not to be test item-related due to the lack of dose-related pattern, lack of
microscopic correlate and/or general overlap and variability in individual values.
After the 28-day dosing-free period, the 1000 mg/kg/day Recovery group males showed
statistically significant higher adrenal gland and thyroid gland weights (absolute and relative
to body weight). In the 1000 mg/kg/day group Recovery group females, there was a
statistically significant higher organ weight of the thyroid/parathyroid gland (relative to body
weight) and statistically significant lower organ weights of the heart (absolute) and liver
(absolute and relative to body weight). A relationship with the test-item was regarded unlikely
based on the lack of statistically significant changes and lack of test item-related microscopic
changes in these organs in Main group rats. Therefore, the differences in organ weights of
Recovery group animals in the high dose group compared to concurrent controls were
attributed to lower final body weights (females) and/or general overlap and variability in
individual values.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations in Main and Recovery group animals.
All of the recorded macroscopic findings were within the range of background gross
observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the mesenteric lymph nodes of
1000 mg/kg/day group females.

An increased incidence and severity of macrophage accumulation was noted in the mesenteric
lymph node of the 1000 mg/kg/day group Main females at minimal or mild degree. After a
28-day treatment-free period, this finding was still present at increased incidence and severity
in 1000 mg/kg/day group females (minimal or mild).
There were no test item-related microscopic observations in males.

A finding of note was observed in one kidney and the urinary bladder of 1000 mg/kg/day
group Main Female No. 87, consisting of diffuse urothelial hyperplasia and concomitant
lymphocytic infiltrate at mild degrees in the kidney and minimal degrees in the urinary
bladder. Since the kidney was unilaterally affected and these findings occurred in a single
Main female at 1000 mg/kg/day, they were regarded as spontaneous and unrelated to
treatment with the test item.
The remainder of the recorded microscopic findings were within the range of background
pathology encountered in rats of this age and strain. There was no test item-related alteration
in the prevalence, severity, or histologic character of those incidental tissue alterations.

Key result

Dose descriptor:

NOAEL

Remarks:

Administration of EC 701-204-9 by once daily oral gavage was well tolerated in rats at dose levels of 100, 300 and1000 mg/kg/day.

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

dermal irritation

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

serum/plasma biochemistry

water consumption and compound intake

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw (total dose)

System:

immune system

Organ:

lymph node

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

In conclusion, administration of EC 701-204-9 by once daily oral gavage was well tolerated in rats at dose levels of 100, 300 and1000 mg/kg/day.

The only relevant test item-related finding was a non-adverse mild exacerbation in incidence and severity of macrophage accumulation in the mesenteric lymph nodes in the 1000 mg/kg/day group females (Main and Recovery).

Based on these results, a No Observed Adverse Effect Level (NOAEL) for EC 701-204-9 of at least 1000 mg/kg/day was established.

Executive summary:

The objective of this study was to determine the potential toxicity of EC 701-204-9, when given orally by gavage for 90 days to Wistar Han rats and to evaluate the potential reversibility of any findings. In addition, a No Observed Adverse Effect Level (NOAEL) was evaluated.

The study design was as follows:

Group n°	Test item ID	Dose level (mg/kg/day)	Dose Volume (mL/kg)	Dose concentration (mg/mL)	Subsets	Number of animals
						Male	Female
1	Control	0 (vehicle)	5	NA	Main	10	10
					Recovery	5	5
2	EC 701-204-9	100	5	20	Main	10	10
3		300	5	60	Main	10	10
4		1000	5	200	Main	10	10
					Recovery	5	5

Chemical analyses of formulations were conducted on Weeks 1, 6 and 13 to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study: clinical signs, functional observation tests, body weights, food consumption, ophthalmology, estrous stage, clinical pathology parameters (hematology, coagulation and clinical chemistry), gross necropsy findings, organ weights, and histopathologic examinations.

Formulations prepared in Weeks 1 and 6 were considered homogeneous at the concentrations tested, and analysis of the accuracy revealed acceptable levels. In Week 13, the mean accuracy of the formulations of Groups 2, 3 and 4 was below target concentrations (i.e. 79%, 83% and 82% of target, respectively). Since no cause for the off-target result was found, additional formulation samples of Week 13 were analysed, which were also below the target concentration (i.e. 81%, 83% and 81% of target, respectively).

As previous results (Weeks 1 and 6) were within specifications and homogeneity was within specifications, and results of Week 13 deviated less than 5% between the first and second analysis, it was decided to accept these results. Based on these results it can be concluded that the Week 13 formulations for the high dose were approximately 3% out of target, which is only a minor deviation from the target range.

In Weeks 1 and 6, animals were dosed with formulations that were within the target concentrations, and in Week 13 this was just below target concentrations (adjusted concentrations based on the average accuracy: 80, 249 and 815 mg/kg/day for respectively Groups 2, 3 and 4). Overall, the results in Weeks 1, 6 and 13 suggest that the formulations were prepared accurately during the course of the study.

No mortality occurred during the study.

At 1000 mg/kg/day, a non-adverse test item-related mild exacerbation in incidence and severity of macrophage accumulation was noted in the mesenteric lymph node of Main females at minimal or mild degree. After a 28 days dosing-free period, this finding was still present at increased incidence and severity (minimal or mild). No other relevant test item related findings were noted at 1000 mg/kg/day.
No test item-related findings were observed in males and females at 100 and 300 mg/kg/day, and in males at 1000 mg/kg/day.

In conclusion, administration of EC 701-204-9 by once daily oral gavage was well tolerated in rats at dose levels of 100, 300 and 1000 mg/kg/day. The only relevant test item-related finding was a non-adverse mild exacerbation in incidence and severity of macrophage accumulation in the mesenteric lymph nodes in the 1000 mg/kg/day group females (Main and Recovery).

Based on these results, a No Observed Adverse Effect Level (NOAEL) for EC 701-204-9 of at least 1000 mg/kg/day was established.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Justification for classification or non-classification

Several repeat dose toxicity studies are available for the registered substance. No adverse effect was observed at any dose level tested. The NOAEL is 1000 mg/L (the highest dose tested).