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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Jan 2016 to 30 Jan 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Chemical Substances Control Law 1973, Notification of Mar. 31 2011 by MHLW (0331 No.7), METI (H23.03.29 SeiKyoku No. 5) and MOE (No. 110331009)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(2Z)-3-({2-[(8E)-9-(1,3-dioxo-7-pentyl-1,3,3a,4,7,7a-hexahydro-2-benzofuran-4-yl)non-8-enamido]ethyl}({2-[(9E)-octadec-9-enamido]ethyl})carbamoyl)prop-2-enoic acid; (2Z)-3-[bis({2-[(8E)-9-(1,3-dioxo-7-pentyl-1,3,3a,4,7,7a-hexahydro-2-benzofuran-4-yl)non-8-enamido]ethyl})carbamoyl]prop-2-enoic acid; (2Z)-3-[bis({2-[(9E)-octadec-9-enamido]ethyl})carbamoyl]prop-2-enoic acid

EC Number:

800-430-6

Cas Number:

1419212-76-2

Molecular formula:

ca. C44H75N3O5 - C48H81N305

IUPAC Name:

(2Z)-3-({2-[(8E)-9-(1,3-dioxo-7-pentyl-1,3,3a,4,7,7a-hexahydro-2-benzofuran-4-yl)non-8-enamido]ethyl}({2-[(9E)-octadec-9-enamido]ethyl})carbamoyl)prop-2-enoic acid; (2Z)-3-[bis({2-[(8E)-9-(1,3-dioxo-7-pentyl-1,3,3a,4,7,7a-hexahydro-2-benzofuran-4-yl)non-8-enamido]ethyl})carbamoyl]prop-2-enoic acid; (2Z)-3-[bis({2-[(9E)-octadec-9-enamido]ethyl})carbamoyl]prop-2-enoic acid

Test material form:

other: paste

Details on test material:

Identification: Reaction products of tall oil fatty acids with diethylenetriamine and maleic anhydride
Appearance: Dark brown viscous liquid/paste
Purity/Composition: UVCB
Test item storage: At room temperature

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl:WI(Han) Recognized by international guidelines as the recommended test system

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: ca 6 weeks
- Weight at study initiation: males 124-134 g; females 111-130 g
- Fasting period before study: none
- Housing:Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material and paper as cage-enrichment
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40-70%
- Air changes (per hr): at least 10/hr
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 March 2016 To: 14 June 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. Adjustment was made for specific gravity/density of the test item and vehicle. No correction was made for purity/composition of the test item

VEHICLE
- Justification for use and choice of vehicle : based on labortory trials
- Amount of vehicle (if gavage): 5 mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate samples were taken from the formulations (0, 100, 300 and 1000 mg/kg bw on day 1, in week 3 and week 13). For determination of accuracy, samples were taken at middle position (50% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the formulations. For determination of stability, additional samples were taken at 50% height and stored at room temperature under normal laboratory light conditions for 6 hours.
Samples were diluted with tetrahydrofuran and further diluted (to fall within the calibration range) to a solution of 0.1% (v/v) formic acid in 50/50 (v/v) tetrahydrofuran/water

UPLC-MS
Instrument Acquity UPLC system (Waters, Milford, MA, USA)
Detector Xevo TQ-S mass spectrometer (Waters)
Column Acquity UPLC BEH C8, 50 mm x 2.1 mm i.d., dp =1.7 µm (Waters)
Column temperature 40°C ± 1°C
Injection volume 5 µL
Mobile phase 0.1% formic acid in 90/10 (v/v) acetonitrile/water

Flow 0.6 mL/min
MS detection
Ionisation source ESI+
Cone voltage 20 V
Collision energy 30 eV
Quantitation sum of m/z 628.5 -> m/z 306 and m/z 630.4 --> m/z 308

Calibration (6 concentration in THF at final concentrations of 5 - 100 µg/L): linear regression (r > 0.99) based on 4 of 6 data points (2 datapoints outside 85-115% of nominal)
QC samples: 1 mg/g 90-108% of nominal, 200 mg/g 93-106% of nominal
Accuracy: 0 mg/kg bw: ND ; 100 mg/kg bw 88-99% of nominal; 300 mg/kg bw: 94-104% of nominal; 1000 mg/kg bw: 87-107% of nominal
homogeneity: 100 mg/kg bw: coefficient of variation 2.2-4.8%; 1000 mg/kg bw coefficient of variation: 2.4-4.6%
Stability over 6 hours: 100 mg/kg bw: relative difference 41%; 1000 mg/kg bw relative difference 7.3%
Stability over 8 days (in refrigerator): not measured because QC samples were average 79 and 69% of nominal (thus outside the acceptable range of 85-115% of nominal)

Duration of treatment / exposure:

at least 90 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

measured concentrations within 85-115% of nominal

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

measured concentrations within 85-115% of nominal

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

measured concentrations within 85-115% of nominal

No. of animals per sex per dose:

10 males + 10 females per dosage group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a dose-range finding study (Charles River 511550):
No toxicologically significant changes related to mortality, clinical signs, body weight, food consumption, macroscopy and organ weights (liver and kidney) were found at 500 and 1000 mg/kg bw (3 female rats/dose exposed by gavage for 14 days).

Positive control:

NA

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (mortality twice daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly in a standard arena

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION : No (checked regularly)

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-test and in week 13
- Dose groups that were examined: pre-test all, week 13 in controls and at 1000 mg/kg bw

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrifice
- Anaesthetic used for blood collection: Yes isoflurane
- Animals fasted: Yes during 25 hours
- How many animals: all
- Parameters checked: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice
- Anaesthetic used for blood collection: Yes isoflurane
- Animals fasted: Yes during 25 hours
- How many animals: all
- Parameters checked: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 13
- Dose groups that were examined: all (5/sex/group, first cage)
- Battery of functions tested: hearing ability , pupillary reflex , static righting reflex, fore- and hind-limb grip strength, locomotor activity (recording period: 1-hour measuring total movements and ambulations)

IMMUNOLOGY: No:

Sacrifice and pathology:

Animals were deprived from food (not water) for 25 hours prior to necropsy

ORGAN WEIGHTS: Yes
from all animals: Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus, Thyroid including parathyroid, Testes, Epididymides, Seminal vesicles including coagulating glands, Prostate, Uterus (including cervix), Ovaries

GROSS PATHOLOGY: Yes , at sacrifice or within 24 hours after found dead
on all animals: Adrenal glands, Aorta, Brain [cerebellum, mid-brain, cortex] (7 levels) , Caecum, Cervix, Clitoral gland, Colon, Duodenum, Epididymides , Eyes with optic nerve [if detectable] and , Harderian gland , Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, Larynx, Lacrimal gland, exorbital, Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, Nasopharynx, Oesophagus, Ovaries, Pancreas, Peyer's patches [jejunum, ileum] if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, Salivary glands - mandibular, sublingual, Sciatic nerve, Seminal vesicles including coagulating gland, Skeletal muscle, Skin , Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes , Thymus, Thyroid including parathyroid [if detectable], Tongue, Trachea, Urinary bladder, Uterus, Vagina, All gross lesions,


HISTOPATHOLOGY: Yes :
on all controls and animals at 1000 mg/kg bw : Adrenal glands, Aorta, Brain [cerebellum, mid-brain, cortex] (7 levels) , Caecum, Cervix, Colon, Duodenum, Epididymides , Eyes with optic nerve [if detectable] and , Harderian gland , Female mammary gland area,, Heart, Ileum, Jejunum, Kidneys, Larynx, Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, Oesophagus, Ovaries, Pancreas, Peyer's patches [jejunum, ileum] if detectable, Pituitary gland, Prostate gland, Rectum, Salivary glands - mandibular, sublingual, Sciatic nerve, Seminal vesicles including coagulating gland, Skin , Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes , Thymus, Thyroid including parathyroid [if detectable], Trachea, Urinary bladder, Uterus, Vagina, All gross lesions
on all animals at 100 and 300 mg/kg bw: liver (Males), thyroid (Females) and all gross lesions

Statistics:

The following statistical methods were used to analyze the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test
based on a pooled variance estimate was applied for the
comparison of the treated groups and the control groups for each sex.
• The Steel-test (was applied if the data could not be
assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test was applied to motor
activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of
significance. Group means were calculated for continuous data and medians were calculated
for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of
exact values for means and pooled variances. Individual values, means and standard
deviations may have been rounded off before printing. Therefore, two groups may display the
same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No toxicological relevant clinical signs during daily observations or abnormalities during weekly arena observations were noted during the observation period.
Salivation noted after dosing for all animals (with a dose increasing trend in incidences) was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response to the test item rather than a sign of systemic toxicity.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and atreated 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.
see attached tables

Mortality:

mortality observed, non-treatment-related

Description (incidence):

A female treated at 1000 mg/kg (no. 72) was found dead on Day 88. During the in-life phase this animal showed salivation and alopecia. No effect was noted in body weight.
Macroscopic examination revealed watery-cloudy contents of the thoracic cavity and a beginning autolysis.
No further mortality occurred during the study period.
A female treated at 1000 mg/kg (no. 72) was found dead on Day 88. During the in-life phase this animal showed salivation and alopecia. No effect was noted in body weight.
Macroscopic examination revealed watery-cloudy contents of the thoracic cavity and a beginning autolysis.
No further mortality occurred during the study period.
see attached tables

Body weight and weight changes:

no effects observed

Description (incidence and severity):

a very slight reduction of body weight (gain) in females at 1000 mg/kg bw (not statistically significant), which was not considered toxicologically relevant
No toxicologically relevant changes were observed in body weights and body weight gain over the study period.
see tables as attached

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after correction for body weight were recorded.
see tables as attached

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmology findings were noted that were considered to be related to treatment.The recorded ophthalmology findings were within the range of background findings encountered in rats of this age and strain.
see attached tables

Haematological findings:

no effects observed

Description (incidence and severity):

Haematological parameters of treated rats were considered not to have been affected by treatment.
Any statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
see attached tables

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Main changes not considered to be toxicologically relevant:
Males 1000 mg/kg bw: significantly decreased bilirubin and bile acids
Females 300 and 1000 mg/kg bw: significantly decreased inorganic phosphate
No toxicologically relevant changes were noted in clinical biochemistry parameters.
Slightly lower total bilirubin and bile acids in males at 1000 mg/kg and slightly lower inorganic phosphate in females at 300 and 1000 mg/kg were only seen in one sex and were well within the expected range for these kind of rats. Therefore these changes were not considered toxicologically relevant.
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
see attached tables

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength and motor activity was similar between control and high dose animals.
All groups showed a similar motor activity habituation profile with a decreasing trend inactivity over the duration of the test period.
see attached tables

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related alterations in organ weights.
Only absolute kidney weights of females at 1000 mg/kg were statistically decreased compared to the concurrent controls (-8 %). This was in line with the small decrease in body weight (-4%), which was considered not toxicologically relevant.
see attached tables

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations.
All recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
see attached tables

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Males 1000 mg/kg bw: liver hypertrophy (n=4)
Females 1000 mg/kg bw: thyroid follicular cell hypertrophy (n=6)
Males: controls and 1000 mg/kg bw: hyaline droplets (not relevant for humans)
Test item-related microscopic findings were noted in the liver of males at 1000 mg/kg and thyroid gland of females at 1000 mg/kg.
Summary Test Item-Related Microscopic Findings
Males
Dose level (mg/kg/day): 0 100 300 1000

LIVER a 10 10 10 10
Hepatocellular hypertrophy
Minimal 0 0 0 4
a = Number of organs examined from each group.
Hepatocellular hypertrophy was present in 4/10 males at 1000 mg/kg (minimal) and absent in all other groups.
Summary Test Item-Related Microscopic Findings
Females
Dose level (mg/kg/day): 0 100 300 1000b

THYROID GLAND a 10 10 10 10
Hypertrophy
Minimal 2 0 1 5
Slight 0 0 0 1
a = Number of organs examined from each group.
b = Including Female 72, 88 testing days, no findings in the thyroid gland.
An increased incidence and/or severity of follicular cell hypertrophy in thyroid gland, was present in 6/10 females at 1000 mg/kg (up to slight), compared to minimal degrees in 2/10 at 0 mg/kg, 0/10 at 100 mg/kg and 1/10 at 300 mg/kg.
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. (This includes the observed hyaline droplets of the male kidneys in groups 1 and 4).
see attached tables

Histopathological findings: neoplastic:

not examined

Details on results:

No treatment related adverse effects were noted. The minimal hepatocellular hypertrophy in the liver of males at 1000 mg/kg/day in the absence of any other indicator of hepatocellular toxicity was not considered adverse (Hall et al, 2012).
The incidences and severities of follicular cell hypertrophy of the thyroid gland recorded for females at 0 mg/kg/day and at 300 mg/kg/day were within background pathology for rats of this age and strain. The increase in incidence and/or severity (up to slight degree) of females at 1000 mg/kg/day was regarded to be an adaptive change (Greaves, 2007) and considered non-adverse at the incidences and low severities recorded and absence of any other treatment-related finding in females.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment related effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

RESULTS

For further detail on summary data, see APPENDIX as attached

Dose	0		100		300		1000		Treatment related
Endpoint	M	F	M	F	M	F	M	F
Mortality	0/10	0/10	0/10	0/10	0/10	0/10	0/10	1/10 (NTRE)	No
Clinical signs -Salivation	Wk 8 onwards	Wk 8 onwards	Wk 8 onwards	Wk 8 onwards	Wk 8 onwards	Wk 8 onwards	Wk 4 onwards	Wk 4 onwards	Yes
Body weight (gain)	NTRE								No
Food consumption	NTRE								No
Behavioral effects	NTRE								No
Motoractivity	NTRE								No
Heamatology					RBC↑ (4%)	MCHC↓(2%)			No
Clinical biochemistry			sodium↑(2%)		sodium↑(2%)	iphosphate↓ (13%)	bilirubin↓ (20%) bile acids↓ (39%	phosphate↓ (13%)	Within historical control
Organ weights								Abs Kidneys↓(8%) Rel Kidneys (-4%)	No
Marcoscopy -Uterus with fluid		4/10		3/10		6/10		6/10	No
Histopathology -Hepatocellular Hypertrophy -Thyroid Hypertrophy -Kidney Hyaline droplets	0/10   8/10	2/10	0/10	0/10	0/10   1/10	2/10	4/10   7/10	6/10	Yes (non-adverse)

NTRE= no treatment related effects

↑/↓= significantly increased/decreased at 1% or 5% level (Dunnet test)

% compared to controls

1Analysis of Dose Preparations

1 Accuracy of preparation

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). 

A small response at the retention time of the test item was observed in the chromatograms of the Group 1 formulation prepared for use in Week 13. It was considered not to derive from carry over since no response was obtained in the analytical blanks. This finding was very slight in week 13 (max.1% of the Group 2 response) and in all other formulations of Group 1, no test item was detected.

Homogeneity

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation = 10%). 

Stability

Analysis of Group 4 formulations after storage at room temperature under normal laboratory light conditions for at least 6 hours yielded a relative difference of = 10%. Analysis of Group 2 formulations yielded a relative difference of > 10%. As no loss of test item was observed and because a relative difference of + 41% seems not explainable by the physicochemical properties of the test item or vehicle, results were first accepted.

 

To confirm this result, analysis of Group 2 formulation was performed after in life phase. Analysis of Group 2 formulation after storage at room temperature under normal laboratory light conditions for at least 6 hours yielded a relative difference of = 10%.

 

Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 6 hours.

 

2 Observations

2.1.Mortality

A female treated at 1000 mg/kg (no. 72) was found dead on Day 88. During the in-life phase this animal showed salivation and alopecia. No effect was noted in body weight.

Macroscopic examination revealed watery-cloudy contents of the thoracic cavity and a beginning autolysis.

No further mortality occurred during the study period.

2.2.Clinical Signs

No toxicological relevant clinical signs during daily observations or abnormalities during weekly arena observations were noted during the observation period.

Salivation noted after dosing for all animals (with a dose increasing trend in incidences) was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response to the test item rather than a sign of systemic toxicity. 

Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and atreated 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.

2.3.Functional Observations

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength and motor activity was similar between control and high dose animals.

All groups showed a similar motor activity habituation profile with a decreasing trend inactivity over the duration of the test period.

2.4.Body Weights

No toxicologically relevant changes were observed in body weights and body weight gain over the study period.

2.5.Food Consumption

No toxicologically relevant changes in food consumption before or after correction for body weight were recorded.

2.6.Ophthalmoscopic Examination

No ophthalmology findings were noted that were considered to be related to treatment.The recorded ophthalmology findings were within the range of background findings encountered in rats of this age and strain.

3.Clinical Laboratory Investigations

3.1.Haematology

Haematological parameters of treated rats were considered not to have been affected by treatment.

Any statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

3.2.Clinical Biochemistry

No toxicologically relevant changes were noted in clinical biochemistry parameters.

Slightly lower total bilirubin and bile acids in males at 1000 mg/kg and slightly lower inorganic phosphate in females at 300 and 1000 mg/kg were only seen in one sex and were well within the expected range for these kind of rats. Therefore these changes were not considered toxicologically relevant.

Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

4.Pathology

4.1.Macroscopic Examination

There were no test item-related gross observations.

All recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. 

4.2.Organ Weights

There were no test item-related alterations in organ weights.

Only absolute kidney weights of females at 1000 mg/kg were statistically decreased compared to the concurrent controls (-8 %). This was in line with the small decrease in body weight (-4%), which was considered not toxicologically relevant.

4.3.Microscopic Examination

Test item-related microscopic findings were noted in the liver of males at 1000 mg/kg and thyroid gland of females at 1000 mg/kg.

Summary Test Item-Related Microscopic Findings

	Males
Dose level (mg/kg/day):	0	100	300	1000
LIVER a	10	10	10	10
Hepatocellular hypertrophy
Minimal	0	0	0	4

^a = Number of organs examined from each group.

Hepatocellular hypertrophy was present in 4/10 males at 1000 mg/kg (minimal) and absent in all other groups.

Summary Test Item-Related Microscopic Findings

	Females
Dose level (mg/kg/day):	0	100	300	1000^b
THYROID GLAND a	10	10	10	10
Hypertrophy
Minimal	2	0	1	5
Slight	0	0	0	1

^a = Number of organs examined from each group.

^b = Including Female 72, 88 testing days, no findings in the thyroid gland.

An increased incidence and/or severity of follicular cell hypertrophy in thyroid gland, was present in 6/10 females at 1000 mg/kg (up to slight), compared to minimal degrees in 2/10 at 0 mg/kg, 0/10 at 100 mg/kg and 1/10 at 300 mg/kg.

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. (This includes the observed hyaline droplets of the male kidneys in groups 1 and 4). 

 

Applicant's summary and conclusion

Conclusions:

Rats (10/sex) were exposed to the substance by oral gavage at 0, 100, 300 and 1000 mg/kg bw during 90 days. One female at 1000 mg/kg bw died on day 88. No treatment effects on clinical signs, bodyweight (gain), food consumption, ophtalmoscopy, functional observations, haematology, clinical biochemistry, organ weights, macroscopy and histopathology were found. The NOAEL is therefore 1000 mg/kg bw.

Executive summary:

Study outline

The test item, formulated in propylene glycol, was administered daily for at least 90 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested,

each consisting of 10 males and 10 females.  

Evaluated parameters

Chemical analyses of formulations were conducted during pre-treatment and in Weeks 3 and 13 to assess accuracy, homogeneity and stability over 6 hours at room temperature under normal laboratory light conditions and over 8 days in the refrigerator protected from light. Additional analysis was done after the in-life phase to confirm the stability at low dose levels.

The following parameters were evaluated: clinical signs daily; functional observation tests in Weeks 13; body weight and food consumption weekly; ophthalmoscopy at pretest and in Week 13; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Results

Formulation analyses confirmed that formulations of test item in propylene glycol were prepared accurately and homogenously, and were stable over at least 6 hours at room temperature. No toxicologically significant changes were noted in clinical appearance, functional observations, body weight, food consumption, ophthalmoscopy, clinical laboratory investigations, macroscopic examination or organ weights in the animals surviving up to the scheduled day of necropsy.  

Microscopic examination revealed minimal hepatocellular hypertrophy in the liver of males at 1000 mg/kg. In the absence of any other indicator of hepatocellular toxicity this finding was not considered adverse. Furthermore an increase in incidence and/or severity (up to slight degree) of follicular cell hypertrophy was recorded in the thyroid gland of females at 1000 mg/kg. This was regarded to be non-adverse at the incidences and severities recorded.

Conclusion

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for Reaction products of tall oil fatty acids with diethylenetriamine and maleic anhydride of at least 1000 mg/kg was established.