Registration Dossier

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Administrative data

Description of key information

A NOEL of 100 mg/kg bw/d was determined for the Ninety day repeated dose oral toxicity study in the rat on the read across substance in an OECD Test Guideline 408 study. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Remarks:
Subchronic Oral Toxicity - Rodent: 90 Day Study” (Adopted 21 September 1998).
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
16th March 2016 to 25th October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with regulatory guidelines and in accordance with the principles for good laboratory practice (GLP).
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
Deviations from the study plan occured however the scientific integrity and validity of the study were unaffected.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Identification : TOFA_TETA_PAA_BADGE_CGE_Adduct
Physical State/Appearance : Yellow viscous liquid
Chemical Name : Fatty acids, tall-oil, reaction products with bisphenol A,
epichlorohydrin, glycidyl tolyl ether and
triethylenetetramine
Purity : UVCB
Batch Number : Ei 2985
Date Received : 29 January 2016
Storage Conditions : ambient temperature in the dark
Expiry Date : 01 December 2018
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 161 to 197g, the females weighed 152 to 182g, and were approximately six to eight weeks old.
-Animals were housed in groups of three or four by sex in solid floor polypropylene cage with stainless steel mesh lids and softwood flake bedding.
DETAILS OF FOOD AND WATER QUALITY:
-The animals were allowed free access to food and water. A pelleted diet was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
-Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels

ENVIRONMENTAL CONDITIONS
-The animals were housed in a single air-conditioned room, the rate of air exchange was at least fifteen air changes per hour.
-The low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.
-The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively.

The diet, drinking water bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

IN-LIFE DATES: From: To: The in-life phase of the study was conducted between 25 March 2016 (first day of treatment) and 24 June 2016 (final day of necropsy).
Route of administration:
oral: gavage
Details on route of administration:
The test item was administered daily, for up to ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Polyethylene glycol 400.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Vehicle:
polyethylene glycol
Remarks:
Polyethylene glycol 400
Details on oral exposure:
VEHICLE
-For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Polyethylene glycol 400 and homogenetity of the test item formulation was determined as part of a previous study.

-Purity: UVCB

-Batch number : Ei 2985
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each test item formulation were taken and analyzed for concentration of TOFA_TETA_PAA_BADGE_CGE_Adduct at Envigo Research Limited, Shardlow, UK,
Analytical Services. The results indicate that the prepared formulations were within 96- 114% of the nominal concentration, confirming the accuracy of the formulation procedure.

Apparatus: Analytical and top-pan balances.

Reagents:
Control vehicle: PEG 400
Tetrahydrofuran: Gradient HPLC grade
Dilution solvent: Tetrahydrofuran


Preparation of calibration standards:
Stock solutions of the test item dilution solvent were prepared for external standard calobration. An aliquot, approx. 0.025 g of test item was exactly weighed into a 200 mL volumetric flask and brought to vilume with dilution solvent to yield a solution with a concentration of 0.125 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration of 0.01 mg/mL.

Preparation of test sample:
The formulation received were diluted with dilution solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with dilution solvent this was then shaken to dissolve. Where neccessary, sample solutions were further diluted with dilution solvent to nachieve the working concentration.

Instrumentation Parameters:
Spectophotometer: Perkin-Elmer Lambda 20
Wavelenght: at 227 nm
Cell path lenght: 1 cm
Reference medium: Tetrahydrofuran

Data evaluation and calculations:
The absorbance response for test item each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. the area response of the peak observed at the characteristic retention time for test item in sample and procedural recovery chromatograms was measured. the concentration of test item was determined using the following equations:

The equation "x" of the test item in an injected sample was calculated by the following equation:
x= y-a / m
Where:
x: concentration of the test item in the injected sample (mg/mL)
y: mean absorbance response of the test item in the injected sample (counts)
m: slope of the calibration plot (counts per mg/mL)
a: y-axis intercept (counts)

The concentration of teh test item in the test sample was calculated using the following equation:
c= (x / mass) x F x SG
Where
c: concentration of the test item in the test sample (mg/mL)
x: concentration of the test item in the injected sample solution (mg/mL)
F. Factor dilution factor
SG: Specific Gravity (where applicable) (g/mL)
Mass: Mass taken (g)

Concentration of Dose Formulations:
For each occasion, freshly prepared test formulations were analyzed. Duplicated samples were analyzed in accordance with the analytical procedure. Sample were disposed of once satisfactory results were achieved.

Major Computerized Systems:
The Computerized Systems that have been used in this project are:
PerkinElmer UV WinLab Enhanced Security Version 6.0.3.0730
Delta BMS

Concentration of dose formulations and conclusion:
The mean concentrations were within applied limits +/- 15 % confirming accurate formulation.

Duration of treatment / exposure:
The test item was administered daily, for up to ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Polyethylene glycol 400.
Frequency of treatment:
The test item was administered daily, for up to ninety consecutive days.
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
This was lowered to 600 mg/kg bw/day after being considered too high from Day 12 for males and Day 13 for females.
This dosage was further reduced to 450 mg/kg bw/day from Day 21 for males and Day 20 for females due to poor body weight performance and mortality during Week 3.
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals male and 10 female animals were used per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose levels of 0 (control), 100, 300 and 1000 mg/kg bw/day were selected for use in this main study. After review of Week 1 data, it was considered that the high dosage of 1000 mg/kg bw/day was too high and this was reduced to 600 mg/kg bw/day from Day 12 for males and Day 13 for females. This dosage was further reduced to 450 mg/kg bw/day from Day 21 for males and Day 20 for females due to poor body weight performance and mortality during Week 3.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized.
Positive control:
Not applicable
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE: YES
- Food consumption was recorded for each cage group at weekly intervals throughout the study.
-Weekly food consumption and efficiency calculated (mg/kg bw/ day)
- Individual and Group mean weekly body weight calculated (g)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): YES
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.


Functional Observations
Prior to the start of treatment and once each week thereafter, all animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.

BEHAVIOURAL ASSESSMENT
- Detailed individual clinical observations were performed for each animal using a purpose built arena. This test was developed from the methods used by Irwin (1968) and Moser et al (1988).
Gait Hyper/Hypothermia
Tremors Skin color
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behavior Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation



OPHTHALMOSCOPIC EXAMINATION: Yes
- The eyes of all control and high dose animals were examined pre-treatment and before termination of treatment.
The eyes of all control and high dose animals were examined pre-treatment and before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye, pupillary and corneal blink reflex. Following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.

HAEMATOLOGY: Yes
- Hematological and blood chemical investigations were performed on all surviving animals from each test and control group at the end of the study
- Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91.
-Animals were not fasted prior to sampling.
In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on all surviving animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling.

- Hemoglobin (Hb)
- Erythrocyte count (RBC)
- Hematocrit (Hct)
- Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)

Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)


Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed

Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).


Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.) Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation) Creatinine (Creat)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++)




URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Prior to the start of treatment and once each week thereafter, all animals were observed for
signs of functional/behavioral toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different
stimuli.
- Battery of functions tested: Motor activity, forelimb/hindlimb grip strength, sensory reactivity



OTHER:
ORGAN WEIGHTS:
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals Ovaries
Brain Spleen
Epididymides Testes
Heart Thymus
Kidneys Uterus
Liver


HISTOPATHOLOGY:
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals Ovaries
Aorta (thoracic) Pancreas
Bone & bone marrow (femur including stifle joint)• Pituitary
Bone & bone marrow (sternum) Prostate
Brain (including cerebrum, cerebellum and pons) Rectum
Caecum Salivary glands (submaxillary)
Colon Sciatic nerve
Duodenum Seminal vesicles
Epididymides ♦ Skin (hind limb)
Esophagus Spinal cord (cervical, mid-thoracic
Eyes* and lumbar)
Gross lesions Spleen
Heart Stomach
Ileum (including Peyer’s patches) Testes ♦
Jejunum Thymus
Kidneys Thyroid/Parathyroid
Liver Tongue•
Lungs (with bronchi) # Trachea
Lymph nodes (mandibular and mesenteric) Urinary bladder
Mammary glands Uterus (with cervix)
Muscle (skeletal)• Vagina

* Eyes fixed in Davidson’s fluid
♦ Preserved in Modified Davidson’s fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative
• Retained only and not processed


All tissues were dispatched to the Test Site (Envigo CRS Limited, Eye, Suffolk, IP23 7PX) for processing (Principal Investigator: J Schofield). All tissues from control and 450 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed. Following the results of the examination of tissues from control and high dose group animals, histology processing and histopathological examination of mesenteric lymph nodes were extended to include animals from the low and intermediate dose groups.


PATHOLOGY
Microscopic examination was conducted by the Study Pathologist (W Henderson). A peer review of the findings observed was conducted by V Mowat at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS, UK.
Sacrifice and pathology:
On completion of the dosing period all surviving animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Other examinations:
Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail 1979).

Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests

The following parameters were observed:
Grasp response Touch escape
Vocalization Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect
the significance of intergroup differences from control; statistical significance was achieved
at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry,
Urinalysis (Volume and Specific Gravity), Absolute Organ Weights, Body Weight-Relative
Organ Weights.
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module
as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method.
The homogeneity of variance from mean values was analyzed using Bartlett’s test.
Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with
appropriate covariates. Any transformed data were analyzed to find the lowest treatment
level that showed a significant effect using the Williams Test for parametric data or the
Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity
of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel
(non-parametric) test to determine significant difference from the control group. Where the
data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test
(parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
For surviving animals, at 1000/600/450 mg/kg bw/day, episodes of increased post-doing
salivation and noisy respiration were apparent for both sexes, the incidence tended to be
higher among these animals when they were receiving a dosage of 1000 mg/kg bw/day. One
male (No. 64) showed decreased respiration rate during Day 2, when receiving 1000 mg/kg
bw/day, while another male (No. 64) showed labored respiration during Days 26‑29,
decreased respiration rate during Days 26‑27 and diarrhea during Days 26‑27. Two females
(No.s 71 and 73) showed hunched posture on Day 84.
At 300 mg/kg bw/day, seven males and five females showed episodes of noisy respiration
during the study and episodes of increased post-doing salivation were apparent for all animals
at this dosage.
At 100 mg/kg bw/day, noisy respiration was apparent for two females and isolated instances
of increased post-dosing salivation were also apparent for one male and four females at this
dosage.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were three unscheduled deaths on the study, all occurring in animals from the highest
dosage group.
Male number 66, receiving 1000/600 mg/kg bw/day, was observed to have piloerection,
diarrhoea and lethargy on Day 11, hunched posture on Days 11-12, noisy respiration on
Days 4, 9-11 and 14-19 and labored and decreased respiration on Day 20. Increased post
(and occasionally pre-) dosing salivation was also observed on Days 6, 7 13-17, 19 and 20.
In view of the declining condition of the animal, it was killed for animal welfare reasons
considerations on Day 20 of the study. Necropsy revealed pale lungs and gaseous distension
in the stomach, jejunum, duodenum, ileum, caecum and colon.
Female number 75, receiving 1000/600 mg/kg bw/day, was killed on Day 20 after showing
hunched posture, distended abdomen and decreased, noisy, labored and gasping respiration.
Previously this female had showed noisy respiration on Days 3, 8-11 and 13-20. Increased
post-dosing salivation was also observed during Days 5, 10, 14-17 and 20. Necropsy
revealed a dark liver and gaseous distension of the stomach, duodenum, jejunum, and
caecum, which was also enlarged.
Male number 69, receiving 1000/600/450 mg/kg bw/day, was observed to have decreased
respiration during Days 64-65 and noisy respiration during Days 12, 22-23, 37-39 51, 54-58
and 66-71. This animal was killed for animal welfare considerations on Day 71. Increased
post-dosing salivation was also observed during Days 6, 8, 15-17, 19, 21-23, 25-26, 29, 32
34-36, 40-42, 45-47, 49-50, 52-53, 55-57, 59, 61-63 and 67-70. Necropsy findings were
restricted to gaseous distension of the caecum.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, statistically significant lower body weight gain for males was
apparent during the first week of treatment, compared to control. Mean body weight gain
remained statistically significant lower than control during the second week of treatment,
during which the dosage was decreased to 600 mg/kg bw/day. Following the lowering of the
dosage to 600 mg/kg bw/day, mean body weight gain remained statistically significant lower
than control during the second week body of treatment. Thereafter body weight gains were
generally similar to control to termination (the dosage received was reduced to 450 mg/kg
bw/day during after Week 3), although overall weight gain from the start of the study was
clearly lower than control at the end of the study.
At 300 mg/kg bw/day, statistically significant lower body weight gain was apparent for males
during the first week of treatment, compared to control. Although no further statistically
significant differences from control were apparent, subsequent body weight gains tended to
be lower than control and overall weight gain at the end of the study was clearly lower than
control.
At 100 mg/kg bw/day, statistically significant lower body weight gain of males was apparent
during the first week of treatment, compared to control. Subsequent body weight gains were
considered to be similar to control and overall body weight gain were only slightly lower than
control at the end of the study.
There were no statistically significant differences in body weight gain for females at all
dosage throughout the study. At 1000 mg/kg bw/day, there was a suggestion of lower body
weight during the first week of treatment but differences from control did not attain statistical
significance and may reflect normal biological variation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Group mean weekly food consumptions are given in Table 8 and are presented graphically in
Figure 3 and Figure 4. Weekly food efficiencies are given in Table 9.
At 300 and 1000/600/450 mg/kg bw/day, food consumption for males was lower than control
throughout the study. At 100 mg/kg bw/day food intake for males was slightly lower than
control during the first four weeks of the study, compared with control. Subsequent food
consumption did not show any consistent differences from control that indicated an adverse
effect of treatment.
At 1000 mg/kg bw/day, there was a suggestion of lower food consumption for females during
the first week of treatment, compared with control. Subsequent food consumption for the
remainder of the study appeared unaffected by treatment.


At 100 and 300 mg/kg bw/day, food consumption for females appeared unaffected by
treatment throughout the study.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, food conversion efficiency was lower than control during the first
two weeks of the study. There was considered to be no clear effect of treatment on food
conversion efficiency following the lowering of the high dosage.
Food conversion efficiency appeared unaffected by treatment for both sexes at 100 and
300 mg/kg bw/day and females at 1000/600/450 mg/kg bw/day.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There was no observed effect of treatment on water consumption for either sex at 100, 300 or
1000/600/450 mg/kg bw/day.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Opthalmoscopic examination of animals of both sexes from the control and 1000/600/
450 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any
treatment-related differences.
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Weekly assessment of the animals in a standard arena did not reveal any obvious effects of
treatment on behavior at 100, 300 or 1000/600/450 mg/kg bw/day.
Both sexes at 1000/600/450 mg/kg bw/day and females at 300 mg/kg bw/day showed
instances of noisy respiration during these assessments and one male at 1000 mg/kg bw/day
also showed decreased respiration. However, these findings were consistent with the general
clinical signs apparent for animals at these dosages during the study
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The incidence, type and distribution of macroscopic findings observed at terminal necropsy of surviving animals did not indicate any effect of treatment at dosages of 100, 300 or 1000/600/450 mg/kg bw/day.
At 1000/600/450 mg/kg bw/day, lower absolute and higher body weight-relative mean liver weights for males attained statistical significance compared to control. For this organ; relative weights are generally considered to be the most accurate indicator of toxicological effect for this organ and all individual relative values for these treated males exceeded the historical control range, however so did 8/10 individual control values. For females at 300 and 1000/600/450 mg/kg bw/day, higher absolute and body weight relative mean liver weights also attained statistical significance compared with control but mean values showed no dosage relationship. The majority of individual values for these treated females exceeded the historical control range but the majority of control values also exceeded the historical control. While a slight underlying effect of treatment for this organ cannot be discounted, in the absence of any supporting histopathological change these differences were considered not to represent an adverse effect.

At 300 and 1000/600/450 mg/kg bw/day, higher absolute and body weight relative mean kidney weights for females attained statistical significance compared to control. Again, for this organ; relative weights are generally considered to be the most accurate indicator of toxicological effect for this organ; 5/10 and 7/9 individual relative values at 300 and 1000/600/450 mg/kg bw/day exceeded the historical control range but 3/10 individual control values also exceeded the historical range. In the absence of any supporting histopathological these findings were considered to be of little toxicological significance and not to represent
an adverse effect of treatment.

At 300 mg/kg bw/day lower absolute and body weight relative mean kidney weights for males attained statistical significance compared with control. The majority of individual control values exceed the historical control range compared to only three at 300 mg/kg bw/day and this finding appears to reflect high control values rather than any effect of treatment. There was no dosage relationship with the group mean values at 1000/600/ 450 mg/kg bw/day being higher than at 300 mg/kg bw/day and in the absence of any supporting histopathological change this finding was considered to be incidental and unrelated to treatment.

For males at all dosages, higher absolute and body weight relative mean adrenal weights attained statistical significance compared with control but absolute values showed no consistent dosage relationship. For absolute adrenal weights 6/9, 5/10 and 4/8 individual values at 100, 300 and 1000/600/450 mg/kg bw/day exceeded the historical control range compared to 3/10 values in the control group. For body weight relative adrenal weights 7/9, 9/10 and 7/8 individual values at 100, 300 and 1000/600/450 mg/kg bw/day exceeded the historical control range compared to 2/10 values in the control group, however relative valuesat 300 and 1000/600/450 mg/kg bw/day were probably adversely influenced by effects on body weight at 300 and 1000/600/450 mg/kg bw/day. Overall, in the absence of any supporting histopathological changes, the differences from control for adrenal weights were considered to be of little toxicological significance and not to represent an adverse effect of treatment.
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Mesenteric Lymph Node: Histiocytosis, mainly evident as histiocyte aggregates in the sinusoids, was present in the mesenteric lymph node of 8/10 males and 10/10 females at 100 mg/kg bw/day, all animals at 300 mg/kg bw/day and all surviving animals at
1000/600/450 mg/kg bw/day. Generally the severity was minimal or mild, although it was moderate in one male at 1000/600/450 mg/kg bw/day, with a dose dependent increase. This change is known to occur in response to the oral administration of some test items. Without further evidence of pathological change, necrosis or abscess formation, it is considered not to affect the functionality of the lymph node and generally considered to be non-adverse Erythrocytosis/erythrophagocytosis was also present in all surviving males at 1000/600/ 450 mg/kg bw/day. This finding is of unknown significance but, in combination with the histiocytosis it may indicate further change and possible adversity for animals at this dosage. Microscopic abnormalities detected for the decedent animals at 1000/600/450 mg/kg bw/day did not indicate any obvious cause of death for any animal:
Male 66 - minimal histiocytosis in the mesenteric lymph node.
Male 69 - minimal histiocytosis and erythrocytosis/erythrophagocytosis in the mesenteric Lymph Node, minimal basophilic tubules in the kidneys, minimal thymic atrophy and congestion/haemorrhage (agonal) of the lungs.
Female 75 - congestion/haemorrhage and centrilobular necrosis (agonal) for the liver, minimal chronic inflammation of the heart and increased alveolar macrophages for the lungs.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
Functional Performance Tests
Intergroup differences in results from functional performance tests did not indicate any obvious effect of treatment for either sex at 100, 300 or 1000/600/450 mg/kg bw/day. Males at all dosages showed statistically significant lower overall activity, compared to control, during the assessment of motor activity at the end of the study, however as group mean values showed no dosage relationship, this was considered to be incidental and unrelated to test item exposure. For females, statistically significant lower mean overall activity was apparent at 100 mg/kg bw/day but, in the absence of any similar statistically significant differences from control at higher dosages, this was also considered to be incidental and unrelated to treatment.
Additionally, at 100 mg/kg bw/day, females showed statistically significant higher mean hind limb grip strength, compared to control, during trial 2, but no statistically significant differences were apparent for trials 1 and 3 or for assessments of fore limb grip strength.
There were no statistically significant differences from control apparent for grip strength of males at this dosage or for either sex at 300 or 1000/600/450 mg/kg bw/day and, in isolation, this finding was considered to be incidental and unrelated to treatment.


Sensory Reactivity Assessments
Intergroup differences observed in the scores for sensory reactivity did not indicate any effect of treatment for either sex at 100, 300 or 1000/600/450 mg/kg bw/day.


In-Life Sampling and Analysis
Hematology

There were considered to be no toxicologically significant effects detected in the hematological parameters examined.
At all dosages, mean hematocrit values for males were statistically significant higher than control, however mean values showed no consistent dosage relationship and all individual values for these treated animals were within the historical control range. In the absence of any supporting histopathological change this finding was considered to be incidental and of no toxicological significance.
At 300 and 1000/600/450 mg/kg bw/day, mean corpuscular volumes for males were statistically significantly higher than control, however mean values showed no consistent dosage relationship. All individual values for these treated animals were within the historical control whilst one control values was below the historical control range. In the absence of any supporting histopathological change this finding was considered to be incidental and of no toxicological significance.
At 100 mg/kg bw/day, mean platelet count for males was statistically significantly lower than control. All individual values for these treated animals were within the historical control whilst two control values exceeded the historical control range. In the absence of any statistically significant differences from control at higher dosages, this finding was considered to be incidental and of no toxicological significance.
At 300 and 1000/600/450 mg/kg bw/day, higher mean erythrocyte counts for females attained statistical significance, compared to control, however, mean values showed no dosage relationship and only two individual values at 300 mg/kg bw/day and one individual value at
1000/600/450 mg/kg bw/day exceeded the historical control range. Females at these dosages also showed statistically significantly lower mean corpuscular hemoglobin values compared to control however, all individual values for these treated animals were within the historical control range whilst two individual control values exceeded this historical range.
Additionally, lower mean corpuscular hemoglobin concentrations for females at all dosages attained statistical significance compared to control, but these mean values showed no dosage relationship. All individual values for these treated animals were within the historical control
range whilst three individual control values exceeded this historical range. Overall these findings for erythrocyte parameters, in the absence of any supporting histopathological change, were considered to be incidental and unrelated to treatment.
At 1000/600/450 mg/kg bw/day, mean total leucocyte count for females was statistically significantly higher than control, principally due a statistically significant higher number of lymphocytes. A total of four individual total leucocyte counts and three individual lymphocytes counts for females at 1000/600/450 mg/kg bw/day exceeded the historical control range compared to only one individual total leucocyte count for control females.

Blood Chemistry

There were considered to be no toxicologically significant effects detected in the blood chemical parameters examined.
At 300 and 1000/600/450 mg/kg bw/day, higher mean aspartate aminotransferase and alanine aminotransferase activities for males attained statistical significance compared with control.
All individual values for these treated animals exceeded the historical control range with three control aspartate aminotransferase values and one control aspartate aminotransferase value also exceeding the historical range.
At 300 and 1000/600/450 mg/kg bw/day, lower mean alkaline phosphatase activities for males attained statistical significance compared with control. However, all individual values were within the historical control range, whilst two control values exceeded this historical range. A decrease in this enzyme activity is considered unlikely to be of any toxicological significance and this finding was considered to be incidental and unrelated to treatment.
At all dosages, mean total protein levels for males were statistical significantly lower than control, although mean differences showed no dosage relationship. At 1000/600/450 mg/kg bw/day, two individual values were below the historical control range, however all individual values at 100 and 300 mg/kg bw/day were within the historical range. There were no accompanying statistical significant differences from control apparent for albumin levels, but mean albumin/globulin ratios were statistically significantly higher than control at 300 and 1000/600/450 mg/kg bw/day. At both dosages, one individual value exceeded the historical control range, however one control value also exceeded this historical range and one further control value was below the historical control range.
At 300 and 1000/600/450 mg/kg bw/day, lower mean total cholesterol levels for males attained statistical significance compared with control, however all individual values for control and treated male animals were within the historical control range. For females at all dosages, statistically significantly lower mean total cholesterol levels, compared to control, were also apparent. With the exception of one individual value at 100 mg/kg bw/day, all individual values for treated females were within the historical control range, however five individual females exceeded this historical range. It is considered that the differences in total cholesterol levels apparent for treated females reflect atypically high control values and,
overall, there was considered to be no effect of treatment on total cholesterol levels for either sex.
At all dosages, higher mean chloride levels for males attained statistical significance compared to control, but these mean values showed no dosage relationship. Only one individual value at 100 mg/kg bw/day and another at 300 mg/kg bw/day exceeded the historical control and all values at 1000/600/450 mg/kg bw/day were within this historical range. At 300 and 1000/600/450 mg/kg bw/day, higher mean potassium levels for males attained statistical significance compared to control, however, mean differences showed no dosage relationship and only one individual value at 300 mg/kg bw/day exceeded the historical control range. Additionally, at 100 mg/kg bw/day, mean sodium levels for males were statistically significantly higher than control but all individual values for these treated animals were within the historical control range. Collectively, these differences from control for male blood electrolyte levels were considered not to indicate any effect of treatment and were considered to be of no toxicological significance.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
The NOAEL for this study was considered to be 100 mg/kg bw/day. At this dosage, males at 100 mg/kg showed lower body weight gain and food intake. The designation of the NOAEL has been driven by the reduction in male body weight.
Sex:
male
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Critical effects observed:
not specified

DISCUSSION:

Treatment of males at 1000 mg/kg bw/day was associated with lower body weight gain, lower food consumption and inferior food conversion efficiency, compared to control, for the first week of the study, which persisted to the second week of the study despite the lowering of the dosage to 600 mg/kg bw/day. Although mean body weight gain during the third week of the study was similar to control, food consumption remained lower and one male and one female were killed due to animal welfare considerations during this third week, necessitating the lowering of this dosage to 450 mg/kg bw/day. Thereafter, body weight gains were generally similar to control although food consumption remained lower throughout the study, however there was a further male decedent on Day 71, due to clinical condition and notable

body weight loss. Overall body weight gain from the start of the study remained notably lower than control. In contrast to males, treatment of females at 1000/600/450 mg/kg bw/day was not associated with any marked effect on body weight gain, food consumption or food conversion efficiency, although there was a suggestion of lower body weight gain and food consumption during the first week of the study, when animals received mainly 1000 mg/kg bw/day.

Clinical signs for surviving animals at 1000/600/450 mg/kg bw/day, included episodes of increased post-doing salivation and noisy respiration for both sexes, with the incidence tending to be higher when animals were receiving a dosage of 1000 or 600 mg/kg bw/day. Sporadic instances of hunched posture, decreased respiration rate, labored respiration, and diarrhea were observed occasionally for these animals during the study. Similar clinical signs, generally to a greater extent, were apparent for the decedent animals. The incidences of noisy respiration apparent during this study may indicate that some of the findings observed may, in part, be due to an irritant effect of the test item in combination with slight aspiration of test item formulation during the dosing procedure rather than just systemic toxicity. However microscopic findings apparent during the study did not indicate any clear irritant effect of the test item during the study and necropsy findings for the decedent animals did not indicate any obvious underlying cause for the observed decline in clinical condition.

There were no mortalities at lower dosages of 100 and 300 mg/kg bw/day. At 300 mg/kg bw/day, body weight gain was statistically significantly lower than control during the first week. Subsequent body weight gains tended to be lower than control, without attaining statistical significance and overall weight gain to termination was clearly lower than control. Food consumption for males at this dosage was also lower than control throughout the study. For males at 100 mg/kg bw/day, lower body weight gain was apparent for the first week of the study but subsequent body weight gains were similar to control and overall body weight gain was only slightly lower than control at termination. Food consumption at this dosage was lower than control during the first four weeks of treatment. For females at 100 and

300 mg/kg bw/day, there was no obvious effect of treatment on body weight gain or food consumption.

Clinical signs at lower dosages consisted of episodes of noisy respiration for two females at 100 mg/kg bw/day and seven males and five females at 300 mg/kg bw/day and incidences of increased post-dosing salivation for one male and four females at 100 mg/kg bw/day and all animals at 300 mg/kg bw/day.

Assessment of blood chemistry parameters revealed higher mean aspartate aminotransferase and alanine aminotransferase activities, compared to control, for males at 300 or 1000/600/450 mg/kg bw/day. These findings were contradicted by lower mean alkaline phosphatase activities for males at these dosages but, this finding was considered incidental and unrelated to treatment. The increase for aspartate aminotransferase and alanine aminotransferase was supported by higher liver weights for males at 1000/600/450 mg/kg bw/day, however there was no associated histopathological change apparent at microscopic evaluation. Additionally similar increased liver weights were apparent for females at 300 and 1000/600/450 mg/kg bw/day without any corresponding increase in aspartate aminotransferase and alanine aminotransferase activities. While the higher mean aspartate aminotransferase and alanine aminotransferase activities observed for males at 300 or 1000/600/450 mg/kg bw/day may indicate some change to the liver, at the level observed this finding was considered not to represent an adverse effect of treatment.

At all dosages, mean total protein levels for males were lower than control, but mean differences showed no dosage relationship. There were no accompanying statistical significant differences from control apparent for albumin levels, but mean albumin/globulin ratios were statistically significantly higher than control at 300 and 1000/600/450 mg/kg bw/day. While it is possible that these parameters may have been influenced by changes in liver metabolism, an association with treatment appears unlikely and, at the level observed, would not represent an adverse effect of treatment.

Macroscopic findings at terminal necropsy did not indicate any effect of treatment at dosages of 100, 300 or 1000/600/450 mg/kg bw/day. Statistically significant differences from control were apparent for a small number of organs, but, in the absence of supporting microscopic change, were considered insufficient to represent an adverse effect.

For males at 1000/600/450 mg/kg bw/day, lower absolute and higher body weight-relative mean liver weights, compared to control, were observed. For females at 300 and 1000/600/450 mg/kg bw/day, higher absolute and body weight relative mean liver weights were also apparent but mean values showed no dosage relationship. While a slight underlying effect of treatment for this organ cannot be discounted, in the absence of any supporting histopathological change, these differences clearly did not represent an adverse effect of treatment.

At 300 and 1000/600/450 mg/kg bw/day, higher absolute and body weight relative mean kidney weights compared to control, were apparent. Additionally, for males at 300 mg/kg bw/day absolute and body weight relative mean kidney weights were lower than control, but this finding appeared to reflect high control values rather than any effect of treatment. In the absence of any supporting histopathological change, the differences from control for females kidney weights were considered to be of little toxicological significance and not to represent an adverse effect of treatment.

For males at all dosages, higher absolute and body weight relative mean adrenal weights compared with control were observed, but absolute values showed no consistent dosage relationship. While body weight relative values did show a dosage relationship, they were probably adversely influenced by effects on terminal body weight, at 300 and 1000/600/ 450 mg/kg bw/day. Overall, in the absence of any supporting histopathological changes, the differences from control for adrenal weights were considered to be of little toxicological

Histopathological changes detected at microscopic evaluation of the tissues considered to be related to treatment were restricted to finding for the mesenteric lymph node. At all dosages, histiocytosis, mainly evident as histiocyte aggregates in the sinusoids, was observed for both sexes, generally at a minimal or mild severity. This change is known to occur in response to the oral administration of some test item and, in isolation, without further evidence of pathological change, necrosis or abscess formation, is considered not to affect the functionality of the lymph node and therefore is generally considered to be non-adverse in nature. However, for males at 1000/600/450 mg/kg bw/day, erythrocytosis/ erythrophagocytosis was also present and while this finding is of unknown significance, in combination with the histiocytosis, it may indicate further change and possible adversity for animals at this dosage. This finding was therefore considered to preclude this dosage from representing a No Observed Adverse Effect level (NOAEL).

While there were no histopathological changes that precluded a dosage of 300 mg/kg bw/day from being regarded as being a NOAEL, effects were apparent for male body weight and food consumption. While these effects were generally to a lesser extent than observed for males at 1000/600/450 mg/kg bw/day, overall body weight gain for males at 300 mg/kg bw/day was only slightly better than observed at the highest dosage. The designation of this dosage as a NOAEL for males is equivocal and therefore the NOAEL for this study is considered to be 100 mg/kg bw/day. At this dosage, males at 100 mg/kg showed lower body weight gain during the first week and lower food intake during the first four weeks of the study, but by the end of the study overall body weight gain was only slightly lower than

control. It should be noted that the designation of the NOAEL has been driven by male toxicity rather than effects for the female and the NOAEL for the female would be at least 300 mg/kg bw/day and possibly higher.

Conclusions:
Based on the results of this study, the No Observed Adverse Effect Level for the rat following 90 consecutive days of treatment with TOFA_TETA_PAA_BADGE_CGE is considered to be 100 mg/kg bw/day.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity of the test item and is compatible with the following regulatory guidelines:

The OECD Guidelines for Testing of Chemicals No. 408 "Subchronic Oral Toxicity - Rodent: 90 Day Study” (Adopted 21 September 1998). This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by oral gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at intended dosages of 100, 300 and 1000 mg/kg bw/day over a period of ninety consecutive days. After review of Week 1 data, it was considered that the high dosage of 1000 mg/kg bw/day was too high and this was reduced to 600 mg/kg bw/day from Day 12 for males and Day 13 for females. This dosage was further reduced to 450 mg/kg bw/day from Day 21 for males and Day 20 for females due to poor body weight performance and mortality during Week 3. A control group of males and females was dosed with the vehicle alone (PEG 400) using a constant dose volume of 4 ml/kg over the same treatment period.

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopy examination was also performed on control group and high dose animals. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. This was extended to include low and intermediate dose animals for the mesenteric lymph nodes.

Results

Mortality

There were three unscheduled deaths on the study, all occurring in animals from the highest dosage group.

One male and one female receiving 1000/600 mg/kg bw/day were killed for animal welfare considerations on their respective Day 20 due to a decline in their clinical condition. Gaseous distension was apparent for the gastrointestinal tract of both animals at necropsy; the male also showed pale lungs and the females a dark liver.

One male receiving 1000/600/450 mg/kg bw/day was killed for animal welfare reasons on Day 71 after showing notable body weight loss. Clinical signs prior to this had included noisy and decreased respiration. Necropsy findings were restricted to gaseous distension of the caecum.

Clinical Observations

For surviving animals, at 1000/600/450 mg/kg bw/day, episodes of increased post-doing salivation and noisy respiration were apparent for both sexes. One male showed an isolated occurrence of decreased respiratory rate, while another male showed occasional instances of labored respiration, decreased respiration rate and diarrhea. Two females also exhibited isolated occurrences of hunched posture.

At 300 mg/kg bw/day, seven males and five females showed episodes of noisy respiration during the study and episodes of increased post-dosing salivation were apparent for all animals at this dosage.

At 100 mg/kg bw/day, noisy respiration was apparent for two females and isolated instances of increased post-dosing salivation were also apparent for one male and four females at this dosage.

Behavioral Assessment

Behavioral Assessments did not indicate any effect of treatment for either sex at 100, 300 or 1000/600/450 mg/kg bw/day.

Functional Performance Tests

Functional performance tests did not indicate any effect of treatment for either sex at 100, 300 or 1000/600/450 mg/kg bw/day.

Sensory Reactivity Assessments

Sensory reactivity assessments did not indicate any effect of treatment for either sex at 100, 300 or 1000/600/450 mg/kg bw/day.

Body Weight

At 1000 mg/kg bw/day, statistically significant lower body weight gain for males was apparent during the first week of treatment, compared to control. Mean body weight gain remained statistically significantly lower than control during the second week of treatment, during which the dosage was decreased to 600 mg/kg bw/day. Thereafter body weight gains were generally similar to control to termination (the dosage received was reduced to 450 mg/kg bw/day after Week 3), although overall weight gain from the start of the study was clearly lower than control at the end of the study.

At 300 mg/kg bw/day, statistically significantly lower body weight gain was apparent for males during the first week of treatment, compared to control. Subsequent body weight gains tended to be slightly lower than control without attaining statistical significance resulting in statistically significant lower overall body weight gain.

At 100 mg/kg bw/day, statistically significant lower body weight gain of males was apparent during the first week of treatment, compared to control but overall body weight gain was only slightly lower than control at the end of the study.

There were no statistically significant differences in body weight gain for females at all dosages throughout the study, although there was a suggestion of lower gain for females receiving 1000 mg/kg bw/day during the first week of the study.

Food Consumption

At 300 and 1000/600/450 mg/kg bw/day, food consumption for males was lower than control throughout the study.

At 100 mg/kg bw/day food intake for males was slightly lower than control during the first four weeks of the study, compared with control.

At 1000 mg/kg bw/day, there was a suggestion of lower food consumption for females during the first week of treatment, compared with control.

At 100 and 300 mg/kg bw/day, food consumption for females was unaffected by treatment throughout the study.

Food Conversion Efficiency

At 1000/600 mg/kg bw/day, food conversion efficiency was lower than control during the first two weeks of the study.

Food conversion efficiency was unaffected by treatment for both sexes at 100 and 300 mg/kg bw/day and females at 1000/600/450 mg/kg bw/day.

Water Consumption

There was no effect of treatment on water consumption for either sex at 100, 300 or 1000/600/450 mg/kg bw/day.

Ophthalmoscopy

Ophthalmoscopy examination of animals receiving 1000/600/450 mg/kg bw/day did not indicate any effect of treatment for either sex.

Hematology

Assessment of hematology parameters did not indicate any effect of treatment for either sex at 100, 300 or 1000/600/450 mg/kg bw/day.

Blood Chemistry

Assessment of blood chemistry parameters did not indicate any adverse effect of treatment for either sex at 100, 300 or 1000/600/450 mg/kg bw/day.

Necropsy

Macroscopic necropsy findings for surviving animals did not indicate any effect of treatment for either sex at 100, 300 or 1000/600/450 mg/kg bw/day.

Organ Weights

Assessment of organ weights did not indicate any adverse effect of treatment for either sex at 100, 300 or 1000/600/450 mg/kg bw/day.

Histopathology

Histiocytosis, mainly evident as histiocyte aggregates in the sinusoids, was present in the mesenteric lymph node of both sexes at 100, 300 and 1000/600/450 mg/kg bw/day.

Erythrocytosis/erythrophagocytosis was also present in the mesenteric lymph node for males at 1000/600/450 mg/kg bw/day.

Conclusion

Based on the results of this study, the No Observed Adverse Effect Level for the rat following 90 consecutive days of treatment with TOFA_TETA_PAA_BADGE_CGE is considered to be 100 mg/kg bw/day.

The study was designed to investigate the systemic toxicity of the test item and is compatible with the following regulatory guidelines:

The OECD Guidelines for Testing of Chemicals No. 408 "Subchronic Oral Toxicity - Rodent: 90 Day Study” (Adopted 21 September 1998).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

The test item was administered by oral gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at intended dosages of 100, 300 and 1000 mg/kg bw/day over a period of ninety consecutive days. After review of Week 1 data, it was considered that the high dosage of 1000 mg/kg bw/day was too high and this was reduced to 600 mg/kg bw/day from Day 12 for males and Day 13 for females. This dosage was further reduced to 450 mg/kg bw/day from Day 21 for males and Day 20 for females due to poor body weight performance and mortality during Week 3. A control group of males and females was dosed with the vehicle alone (PEG 400) using a constant dose volume of 4 ml/kg over the same treatment period. Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopy examination was also performed on control group and high dose animals. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. This was extended to include low and intermediate dose animals for the mesenteric lymph nodes.

Based on the results of this study, the No Observed Adverse Effect Level for the rat following 90 consecutive days of treatment with TOFA_TETA_PAA_BADGE_CGE was considered to be 100 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP study carried out to OECD guideline 408

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

OECD Test Guideline 422

The sub-acute repeated dose oral toxicity of Fatty acids, C18-unsatd., dimers, oligomeric reaction products with tall-oil fatty acids and triethylenetetramine was investigated in rats in a combined repeated dose and reproduction/developmental toxicity screening test conducted according to OECD Test Guideline 422 (Perks, 2013). In the study, groups of male and female Crl:WI(Han) rats (10 animals/group) were administered with the test substance orally by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day. Dose levels were selected based on the results of a 14-day range finding study. No concentration verification results were obtained in the study. Formulations were prepared and used daily for dose administration and dose preparations records showed that the formulations were accurately prepared. In-life data confirmed, indirectly, that a range of dose levels were used. In the absence of analytical conformation the dose levels specified for the main study were considered to be nominal values.

Male rats were dosed once daily for two weeks prior to pairing, during the pairing period and a further two weeks before necropsy. Males were treated for a minimum of 6 weeks prior to necropsy. Female rats were dosed for two weeks prior to pairing, during pairing and until Day 4 post-partum (i.e. approximately 7 weeks of dosing). The females were allowed to litter and rear their offspring to Day 4 post-partum. The following parameters were assessed during the study: clinical observations, body weight, food intake, functional observations and tests, locomotor activity, haematology, clinical chemistry, organ weights, gross pathology and histopathology.

There were no treatment related deaths during the study. One female treated at 1000 mg/kg bw/day was euthanised on Day 1 post-partum, but this death was not considered to be treatment related. While noisy respiration was observed on several occasions during the dosing period in males and females treated at 300 or 1000 mg/kg bw/day, it was considered that this finding was not a direct clinical effect of the test material. Occasionally, mouth rubbing, salivation and/or paddling of the forelimbs were noted in animals from immediately post-dose until the end of the day. The number of animals affected and the duration of the reactions were dose-related. These findings were considered to be a reaction to the taste of the test material and of no toxicological significance.

There was a statistically significant reduction in mean body weight gain over the first week of the dosing period in males treated at 1000 mg/kg bw/day. A lower mean body weight persisted throughout the dosing period and was associated during Week 1 and 2 with a lower mean food consumption (compared to controls). It was considered that these changes did not adversely affect the males as there were no other adverse effects observed, and no effect on behaviour (based on a battery of observational tests) or on mating behaviour. There was a statistically significant reduction in mean body weight gain during the last week of gestation in females treated at 1000 mg/kg bw/day, but in the absence of other effects this transient observation was considered not to be adverse. Group mean locomotor activity data were highly variable. Males in the 1000 mg/kg bw/day dose group showed statistically increased activity compared to the controls, but in the absence of other effects observed during behavioural tests and observations, this was considered unlikely to be of biological significance.

There was a statistically significant increase in alanine aminotransferase levels in males treated at 300 and at 1000 mg/kg bw/day and in females treated at 1000 mg/kg bw/day. There was also an increase in aspartate aminotransferase levels in males and in females treated at 1000 mg/kg bw/day; a finding that may indicate liver changes. There were also slight increases in inorganic phosphate and total cholesterol concentration, achieving a significant dose-response. In the absence of changes in liver weight and microscopic morphological liver changes, these findings were not considered to be adverse. There was a statistically significant reduction in mean urine volume and an increase in specific gravity in males treated at 1000 mg/kg/day, compared with the controls. With only a single measurement and with no visual observations of lower water consumption during the in-life phase, this finding was considered to be incidental.

In males treated at 1000 mg/kg/day, there was a statistically significant decrease in mean heart weight relative to body weight, when compared with the controls. There were no histopathological changes or functional changes (FOBs) and no statistically significant changes in female heart weight. In the mesenteric lymph node, increased histiocyte foci were present in all male and most female rats treated with with the substance at 1000 mg/kg/day. Histiocyte foci were characterised by discrete aggregates of plump eosinophilic macrophages. These changes were considered to be not adverse.

Based on this study, The No-Observed-Adverse-Effect-Level (NOAEL) for the sub-acute repeated dose oral toxicity (general and systemic effects) of the substance in adult male and female rats was considered to be 1000 mg/kg bw/day (i.e. the highest dose tested).

The fatty acids, C18-unsatd., dimers, oligomeric reaction products with tall-oil fatty acids and triethylenetetramine is a precursor of Fatty acids, C16 -18 unsatd., polymers with bisphenol A, butyl glycidyl ether, epichlorohydrin and triethylenetetramine, having a lower molecular weight compared to the crosslinked reaction product, and therefore considered more bioavailable. Thus, although it may therefore bee seen as worst case surrogate, it lacks certain functional gorups and this is why a 90-day oral dosed repeated dose toxicity study is proposed to investigate long term toxicity of fatty acids, C16 -18 unsatd., polymers with bisphenol A, butyl glycidyl ether, epichlorohydrin and triethylenetetramine.

A 90-day oral repeated dose toxicity study in the rat (OECD Test Guideline 408) is proposed to further characterise the repeated dose toxicity of Fatty acids, C16 -18 unsatd., polymers with bisphenol A, butyl glycidyl ether, epichlorohydrin and triethylenetetramine.

Waivers are proposed for repeated dose toxicity tests via the inhalation and dermal routes in accordance with Column 2 of Annex VIII of the REACH Regulation. The repeated dose toxicity of the substance will be adequately elucidated by the oral route. Further testing via the dermal and inhalation routes is not required.

OECD Test Guideline 408 from Read Across Substance

TOFA_TETA_PAA_BADGE_CGE

Introduction

The study was designed to investigate the systemic toxicity of the test item and is compatible with the following regulatory guidelines:

The OECD Guidelines for Testing of Chemicals No. 408 "Subchronic Oral Toxicity - Rodent: 90 Day Study” (Adopted 21 September 1998). This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

The test item was administered by oral gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at intended dosages of 100, 300 and 1000 mg/kg bw/day over a period of ninety consecutive days. After review of Week 1 data, it was considered that the high dosage of 1000 mg/kg bw/day was too high and this was reduced to 600 mg/kg bw/day from Day 12 for males and Day 13 for females. This dosage was further reduced to 450 mg/kg bw/day from Day 21 for males and Day 20 for females due to poor body weight performance and mortality during Week 3. A control group of males and females was dosed with the vehicle alone (PEG 400) using a constant dose volume of 4 ml/kg over the same treatment period.

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopy examination was also performed on control group and high dose animals. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. This was extended to include low and intermediate dose animals for the mesenteric lymph nodes.

Hematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopy examination was also performed on control group and high dose animals. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. This was extended to include low and intermediate dose animals for the mesenteric lymph nodes.

Based on the results of this study, the No Observed Adverse Effect Level for the rat following 90 consecutive days of treatment with TOFA_TETA_PAA_BADGE_CGE was considered to be 100 mg/kg bw/day.


Justification for classification or non-classification

The available data indicate that Fatty acids, C18-unsatd., dimers, oligomeric reaction products with tall-oil fatty acids and triethylenetetramine, a surrogate for fatty acids, C16 -18 unsatd., polymers with bisphenol A, butyl glycidyl ether, epichlorohydrin and triethylenteramine, does not cause target organ toxicity after sub-acute repeated oral doses. Thus, the substance does not meet the criteria for classification for repeated dose toxicity according to Directive 67/548/EEC or Regulation 1272/2008/EC.