Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity - oral: One reliable study is available for this exposure route (Klimisch 1). In the 90 -day oral repeated dose toxicity study in rats performed according to OECD Guideline 408, there were no associated treatment related changes in 100 mg/kg bw/day animals, therefore the NOAEL for systemic toxicity was considered to be at least 100 mg/kg bw/day.

Repeated dose toxicity - dermal: One reliable study is available for this exposure route (Klimisch 1). In the 90-day dermal repeated dose toxicity study in rats performed according to the OECD guideline 411, the substance did not produce systemic toxicity when administered five days per week for 30 and 90 days at doses of 16, 50 and 160 mg/kg/day via dermal route of administration. Therefore, it is concluded that the NOAEL is above 160 mg/kg bw/day.
Repeated dose toxicity - inhalation: No reliable data were available for this exposure route. Therefore no NOAEL for this route of administration was established.

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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-05-10 - 2018-07-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
21 September 1998
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3717
- Expiration date of the lot/batch: 01 December 2019
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature and humidity; used/formulated in light
- Solubility and stability of the test substance in the solvent/vehicle: The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results showed the formulations to be stable for at least 21 days when stored refrigerated. Formulations were therefore prepared every 14 days and stored at approximately 4°C in the dark.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Distilled water.
Species:
rat
Strain:
Wistar
Remarks:
Han™:RccHan™:WIST
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 and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: no data
- Age at study initiation: approximately six weeks old
- Weight at start of treatment: males: 188 to 227 g; females: 149 to 181 g
- Fasting period before study: no
- Housing: The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels.
- Diet (e.g. ad libitum): ad libitum, a pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used
- Water (e.g. ad libitum): ad libitum, mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 8 days during which time their health status was assessed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

IN-LIFE DATES:
From: 19 May 2017 To: 18 August 2017
Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the 7 day range finder toxicity study in rats are believed to be of value in predicting the likely toxicity of the test item to man.
The test item was administered by gavage using a stainless steel cannula attached to a disposable plastic syringe.
Vehicle:
water
Remarks:
distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in distilled water.
Formulations were therefore prepared every 14 days and stored at approximately 4 °C in the dark.

VEHICLE
- Concentration in vehicle: 0, 2, 20 and 40/30/15 mg/mL (Dose level and dose concentration reduced from Day 31 (males) and Day 30 (females) and then further reduced from Day 36 (males) and Day 35 (females))
- Treatment volume: 5 mL/kg. 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYTICAL METHOD: The test item concentration in the test samples was determined by high performance liquid chromatography-mass spectrometry (HPLC-MS) using an external standard technique. The test item gave a chromatographic profile consisting of two peaks.
Stock solutions of the test item in dilution solvent were prepared for external standard calibration. An aliquot, approximately 0.1 g of test item was exactly weighed into a 100 mL volumetric flask and brought the volume with dilution solvent to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solution in dilution solvent with a concentration range of 0.05 mg/mL to 0.25 mg/mL.
On each occasion standard solution derived from two stock standard solutions were used for calculation.
Calibration solutions were injected onto the instrument, at the beginning and end of each sample analysis sequence as a minimum.
To assess the calibration range of the method, a range of standard solutions were prepared in dilution solvent from a stock solution of 1.018 mg/mL by serial dilution covering the concentration range 0.0509 mg/mL to 0.2540 mg/mL.

Results:
- Method validation:
The analytical procedure was successfully validated for test item in distilled water with respect to the specificity of chromatographic analysis, the linearity of detected response, method accuracy and precision.
The specificity of the analytical method was demonstrated by the absence of a peak at the characteristic retention time for test item in the control sample chromatogram.
The calibration data were found to have a quadratic correlation within the calibration range of 0.0509 mg/mL to 0.2540 mg/mL. The R² fit of the calibration curve to the data was 0.994, and was considered to be acceptable.
A mean recovery value of 105% (CV = 3.14%, n=5) was obtained for 1 mg/mL and 96% (CV=5.06%, n=5) was obtained for 100 mg/mL.
The limit of quantification (LOQ) was determined as the lowest standard concentration used during the study.
- Homogeneity and stability of dose formulations:
The homogeneity and stability of test item in distilled water formulations was assessed with respect to the level of concentration at nominal concentrations of 1 mg/mL and 100 mg/mL.
Homogeneity was confirmed at the initial stability time point. The mean analyzed concentration for the nine samples remained within 10% of the initial time zero value and the variation was less than 10%.
- Concentration of dose formulations:
The mean concentrations of test item in test formulations were within applied limits ± 10% (excluding 20 mg/mL during Week 1 analysis), confirming accurate formulation.

Conclusion:
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision.
The homogeneity and stability was confirmed for test item in distilled water formulations at nominal concentrations of 1 mg/mL and 100 mg/mL when stored refrigerated for 12 and 21 days.
The mean concentrations of test item in test formulations analyzeed for the study were generally within ± 10% of nominal concentrations, confirming accurate formulation.

TEST ITEM FORMULATION ANALYSIS:
Samples of test item formulations were taken on four occasions and analyzed for concentration of Jeffamine T-403 at Envigo Research Limited (UK). The results indicate that the prepared formulations were within 93-103% of the nominal concentration.
Duration of treatment / exposure:
90 consecutive days
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle control group (distilled water)
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
intermediate dose group
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
high dose group, from start of treatment until day 31 (males) or day 30 (females)
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
high dose group, from day 31 until day 36 (males), from day 30 until day 35 (females)
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
high dose group, from day 36 (males) or day 35 (females) until end of treatment
No. of animals per sex per dose:
10 males and 10 females per dose group, 80 animals in total
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on the seven day repeated dose oral (gavage) range-finding toxicity study in the rat:
The oral (gavage) administration of Jeffamine T-403 at dose levels of 75, 200 and 300 mg/kg bw/day for a period of up to seven consecutive days, resulted in treatment-related effects in animals of both sexes treated with 300 mg/kg bw/day. The necessary early termination of the 300 mg/kg bw/day dose level excludes this dose level from further investigation. Therefore, dose levels of 0 (Control), 10, 100 and 200 mg/kg bw/day are recommended for use in the planned 90 Day Study.

- Rationale for animal assignment (if not random): 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:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
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.

DETAILED CLINICAL OBSERVATIONS: Yes
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.

BODY WEIGHT: Yes
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:
Food consumption was recorded for each cage group at weekly intervals throughout the study

FOOD EFFICIENCY:
Food conversion efficiency was calculated retrospectively.

WATER CONSUMPTION: Yes
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of all control and treated animals were examined pre-treatment and all control and high dose animals were examined before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye and 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 an ophthalmoscope was performed.

HAEMATOLOGY: Yes
Hematological 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.
The following parameters were examined: 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).

CLINICAL CHEMISTRY: Yes
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.
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 at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. During week 12, functional performance tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.
- Behavioral assessment:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
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
This test was developed from the methods used 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.
- 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

IMMUNOLOGY: No

Sacrifice and pathology:
- Necropsy:
On completion of the dosing period all surviving animals were euthanised 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.

- 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 - preserved in Modified Davidson's fluid, Skin, Esophagus, Spinal cord (cervical, mid-thoracic and lumbar), Eyes - fixed in Davindson's fluid, Gross lesions, Spleen, Heart, Stomach, Ileum (including Peyer’s patches), Testes - preserved in Modified Davidson's fluid, Jejunum, Thymus, Kidneys , Thyroid/Parathyroid, Liver, Tongue - retained only and not processed, Lungs (with bronchi) - Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative, Trachea, Lymph nodes (mandibular and mesenteric) , Urinary bladder, Mammary glands, Uterus (with cervix), Muscle (skeletal) - retained only and not processed, Vagina
Other examinations:
ORGAN WEIGHTS:
The following organs, removed from animals that were euthanised 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
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, 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 treatmentlevel 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).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The observed clinical effects were not considered to be treatment-related. Increased salivation was evident in high dose males on Day 16 and high dose females between Days 9 and 15. Noisy respiration was evident in males from the high dose group between Days 11 and 91 and in females between Days 29 and 88. Instances of noisy respiration were also evident in animals of either sex treated with 100 mg/kg bw/day between Days 50 and 74 for males and between Days 40 and 88 for females and on three occasions in three females treated with 10 mg/kg bw/day.
No such effects were detected in males treated with 10 mg/kg bw/day.
Generalized fur loss was evident in three females treated with 10 mg/kg bw/day and one control female between Days 33 and 89 and Days 50 and 51 (respectively). Observations of this nature are commonly observed in group housed animals and is considered to be incidental.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two males treated with 200 mg/kg bw/day were found dead on Days 16 and 30. Two females treated with 200 mg/kg bw/day were also found dead on Day 23. Following the reduction of the high dose level to 150 mg/kg bw/day, a further male was found dead on Day 35. One male treated with 100 mg/kg bw/day was killed in extremis on Day 83.
The two females treated with 200 mg/kg bw/day that were found dead and one of the males from this treatment group that was found dead on Day 16 did not show any clinical signs prior to death. The male that was found dead on Day 30 showed labored respiration, decreased respiratory rate, prostration and clonic convulsions on Day 30. The male that was found dead on Day 35, only previously showed increased salivation.
The male treated with 100 mg/kg bw/day that was killed in extremis on Day 83 showed noisy/gasping respiration, decreased respiratory rate, prostration and clonic convulsions on Day 83.
There were no further unscheduled deaths.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related changes in body weight or body weight gain.
Males treated with 200 mg/kg bw/day showed a slight reduction in body weight gain during the first week of treatment. Statistical significance was not achieved and recovery was evident thereafter. Following the reduction of the dose level to 150/75 mg/kg bw/day, body weight gain for these males was generally higher than controls, attaining statistical significance (p<0.01) during Week 6.
Females treated with 200 mg/kg bw/day showed a reduction in body weight gain during the first two weeks of treatment. Statistical significance was not achieved and improvement was evident thereafter. However, as a consequence, overall body weight gain for females treated at the high dose group was 16% lower than controls.
No such effects were evident in animals of either sex treated with 100 or 10 mg/kg bw/day.
Males treated with 100 mg/kg bw/day showed a statistically significant reduction (p<0.05) in body weight gain during Week 8. Body weight gains for these males prior to and following this week were comparable to controls, therefore, this intergroup difference was considered to be incidental and of no toxicological importance.No effect on body weight gains was observed for females treated with 100 mg/kg bw/day. No effects on body weight gains were observed for animals of either sex treated with 10 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no treatment-related changes in food consumption.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Slight reductions in food conversion efficiency was evident in animals of either sex treated at the high dose level, however, these generally followed the fluctuations in body weight gain seen in these animals.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There were no treatment-related changes in water consumption. Visual inspection of water bottles did not reveal any inter-group differences.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related changes observed during the ophthalmoscopic examination of animals of both sexes from the control group and surviving high dose group during week 12 of the treatment period.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related changes in hematology. Females from the high dose group showed statistically significant increases (p<0.05) in total leucocyte count, neutrophil and lymphocyte counts. Males from this treatment group also showed a statistically significant increase (p<0.01) in neutrophil count. The majority of individual values for total leucocyte count and neutrophil count and one lymphocyte count were above historical control ranges.
No such effects were evident in animals of either sex treated with 100 or 10 mg/kg bw/day.
Females treated at the high dose level and at 100 mg/kg bw/day showed a statistically significant increase (p<0.05) in mean corpuscular hemoglobin concentration. Females treated with 100 mg/kg bw/day also showed a statistically significant reduction (p<0.05) in platelet count. The majority of individual values were within historical control ranges and in the absence of a true dose related response or any associated histopathological correlates at the high dose level, the intergroup differences were considered not to be of toxicological significance. No such effects were noted in males treated with 100 mg/kg bw/day. No effects were evident in animals of either sex treated with 10 mg/kg bw/day.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects detected in the blood chemical parameters examined.
Females treated at the high dose level and at 100 mg/kg bw/day showed a statistically significant reduction (p<0.01) in urea. A true dose related response was not evident, all of the individual values were within historical control range and no associated histopathological correlates were evident, therefore, the intergroup differences were considered not to be of toxicological importance. Females treated at the high dose level showed a statistically significant reduction (p<0.01) in creatinine. Although the majority of individual values were below historical control range, there were no associated histopathological correlates, therefore the intergroup difference was considered not to be of toxicological importance.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related changes in behavioral assessment, functional performance or sensory activity
- Behavioral assessments: there were no treatment-related changes in behavioral assessments. Instances of noisy respiration were evident in two males and four females at the high dose group during Weeks 4 and 5 (males) and Weeks 4, 6, 8, 10, 11 and 13 (females). Noisy respiration was also evident in two males and one female treated with 100 mg/kg bw/day during Weeks 12 and 13 and during Week 3 (respectively). These correlated with the daily clinical observations seen in these treatment groups. No such effects were evident in animals of either sex treated with 10 mg/kg bw/day.

- Functional performance tests: There were no toxicologically significant changes in functional performance.
Females treated at the high dose level showed a statistically significant reduction (p<0.01) in hind limb grip strength. The intergroup difference was confined to one out of the three tests and in the absence of any clinical signs of neurotoxicity, the intergroup difference was considered not to be of toxicological importance.

- Sensory reactivity assessments: There were no treatment-related changes in sensory reactivity.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no treatment-related effects detected in the organ weights measured. Statistical analysis of the data did not reveal any significant intergroup differences.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant macroscopic abnormalities were detected at terminal necropsy. One of the high dose females that was found dead on Day 23 did not show any macroscopic abnormalities. The other high dose female that was found dead on Day 23 had an enlarged stomach with a thin glandular and non-glandular region, gaseous distension in the stomach, cecum and colon, a dark liver, thymus and uterus and a green discolored mammary gland. The high dose male that was found dead on Day 16 had a dark liver, small seminal vesicles, a thin non-glandular region of the stomach and a hole in the esophagus. The other two high dose males that were found dead had reddened lungs and the male which died on Day 35 also had a dark liver. The male treated with 100 mg/kg bw/day that was killed in extremis on Day 83 had a raised limiting ridge in the stomach and a reddened glandular region of the stomach.

One control male and one male treated with 10 mg/kg bw/day had reddened lungs at necropsy. Such a finding is common in this type of study and was considered unrelated to treatment with the test item. One male treated with the high dose level had a hard and enlarged left seminal vesicle. One female from the high dose group had a reddened liver. In the absence of any associated histopathological correlates these findings were considered not to be of toxicological significance. One female treated with 100 mg/kg bw/day had a pale liver. In isolation, this was considered incidental. One control male had small and flaccid testes. In the absence of treatment, this was considered to be incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related findings during histopathological evaluations.
Premature decedents:
The male from the high dose group that was found dead on Day 16 had a tear in the esophagus (evident at necropsy) and this was considered likely to have been caused by a dosing accident. Inflammatory changes were also evident in the nasopharynx.
One of the females from the high dose group that was found dead on Day 23 had inflammation in the nasopharynx and the cause of death was considered to be reflux of the test item formulation. The other female that was found dead on Day 23 had notable autolysis, however, the cause of death was undetermined.
The male from the high dose group that was found dead on Day 30 had minimal regenerative hyperplasia in the trachea, ulceration in the nasopharynx and minimal increased hematopoiesis in the spleen. The cause of death was considered likely to have been reflux of the test item formulation.
The male from the high dose group that was found dead on Day 35 had minimal regenerative hyperplasia in the trachea and inflammatory changes in the nasopharynx, no other changes were noted and the cause of death was considered to be reflux of the test item formulation.
The male treated with 100 mg/kg bw/day that was killed in extremis on Day 83 had no notable changes and the cause of death was undetermined.
Terminal Necropsy:
There were minor non-specific changes in the high dose group but no consistent changes were evident which indicated a toxicological effect in those animals surviving to the end of the study. Examination of all tissues from 100 mg/kg bw/day animals and the esophagus and trachea from 10 mg/kg bw/day animals indicated no effects related to the administration of the test item.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Details on results:
DISCUSSION
The oral (gavage) administration of Jeffamine T-403 for up to ninety consecutive days, to Wistar rats of both sexes at dose levels of 10, 100 or 200 mg/kg bw/day (reduced to 150 mg/kg bw/day on Day 31 for males and Day 30 for females and then further reduced to 75 mg/kg bw/day on Day 36 for males and Day 35 for females) resulted in the unscheduled death of three males and two females at the high dose group and the early termination of one male treated with 100 mg/kg bw/day.
Of the five unscheduled death, there was no evidence of systemic toxicity. The cause of death for three of these animals was considered to be the indirect partial reflux of gastric contents due gavage dose administration of an irritant material, resulting in the degenerative and inflammatory changes in the upper respiratory tract. Histopathological examination of the premature decedents at the high dose group revealed regenerative hyperplasia in the trachea, ulceration in the nasopharynx and/or inflammatory changes in the nasopharynx. The cause of death in a fourth animal was a hole in the esophagus, which was the result of a physical injury occurring during the dosing procedure. The cause of death for the fifth animal could not be determined due to notable autolysis.

For all sporadic unscheduled deaths there were no associated target organ histopathological changes to suggest that mortality or morbidity was associated with, or a consequence of, systemic toxicity. The evidence of degenerative and inflammatory changes in the upper respiratory tract of these animals supports the conclusion that these deaths were associated with the process of gavage dose administration of an irritant material; the aetiology of the pathology is considered to be gastric reflux. It is recognised that rats do not demonstrate the normal emetic reflex that is seen in humans;, however, the mechanical process of intubation of the oesophagus/stomach with a catheter and subsequent introduction of the test item into the stomach may induce a minor reflux of gastric contents at the time of withdrawal of the catheter. This process of gastric reflux has been reported as an observation in other studies (Damsch, et al, 2011). The authors reported that physical changes to structures such as the upper respiratory tract can take place as a result of gastric reflux. These changes may be sufficient to result in mortality. Respiratory changes prior to death were the most common findings observed for this study. The pathological changes may extend into the nasal structures (Damsch, et al, 2011) which indicate the extent of pathological change that may result from gastric reflux. In a separate study in rats where chronic acid reflux oesophagitis was surgically induced, inflammatory changes in the larynx and pharynx could be identified (Shimazu, et al, 2009). The clinical effects of gastric/oesophageal reflux have been associated with respiratory disease in humans (Molyneux, et al, 2011). Human ingestion of strong irritants such as ammonia (PHE publications, 2015) can result in aspiration of the material resulting in a condition described as acute respiratory distress syndrome. The animals that died on this study generally showed minimal clinical signs of ill-health which suggests that affects leading to mortality were spontaneous and not necessarily related to cumulative toxic effects. This is supported by a lack of consistency with the timing of mortalities on the study and clinical observations. The conclusions that may therefore be drawn from the mortalities on this study are as follows:
i. A combination of factors resulted in the mortalities. The primary cause was considered to be the process of orally intubating a potentially irritant material to rats. This physical process is likely to have resulted in partial reflux of gastric contents on isolated occasions, affecting the upper respiratory tract.
ii. It is likely that the extent of gastric reflux determined the clinical effects observed. In its mildest form there was evidence of post dosing salivation, which progressed to animals with noisy respiration and ultimately animals with severe respiratory distress and morbidity/mortality.
iii. The appearance of the findings was not predictable but was more prevalent at higher concentrations of the test item. The time to appearance of reactions to treatment suggests the animals were demonstrating progressive reactions to the dose administration process.
iv. The lack of clinical effects and subsequent pathology evaluations clearly confirms that there was no underlying systemic toxicity which could have prompted such mortalities.
Isolated and sporadic in nature incidences of increased salivation and noisy respiration were also evident in some of the surviving animals of both sexes treated at the high dose level and to a lesser extent, in a few animals from both sexes treated with 100 mg/kg bw/day. In addition, a very low incidence of noisy respiration at 10 mg/kg bw/day was evident in three females. These findings may also be considered a consequence of the dose administration process. Body weight gain was initially affected at the high dose level in either sex; however, following the reduction of the dose level, improvement was evident thereafter. Food consumption for all treated animals was unaffected.
Hematological investigations revealed changes in white blood cell counts in either sex treated with the high dose level only. Although the majority of individual values were above historical control ranges, no associated histopathological changes were evident at the end of the treatment period. Therefore, these changes may be the delayed result of the initial insult by the test item when the animals were treated with 200 mg/kg bw/day and as no further toxicity was evident at 75 mg/kg bw/day or at 100 mg/kg bw/day, these changes can be regarded as non-adverse.
The clinical signs evident in the male treated with 100 mg/kg bw/day that was euthanized in extremis were the same observations as those seen in one of the premature decedents at the high dose level. Whilst there were no histopathological changes to the upper respiratory tract for this animal the observation of significant respiratory changes prior to termination do lead to the conclusion that morbidity was due to the same cause as that seen amongst decedents at the higher dose initial dose level of 200/150 mg/kg bw/day. However, based on the lack of any significant systemic toxicity observed amongst animals at 100 mg/kg bw/day as observed by the lack of any significant histopathology or blood chemistry/hematology changes plus no significant effects on body weight development or food consumption the No Observed Adverse Effect Level (NOAEL) is considered to be 100 mg/kg bw /day. The higher dose level for this study could not be clearly justified as a suitable NOAEL since the reduction of dose level prevents the accurate assignment of treatment-related effects to the potential cooresponding dose level.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Critical effects observed:
not specified
Conclusions:
The oral (gavage) administration of Jeffamine T-403 for up to ninety consecutive days, to Wistar rats of both sexes at dose levels of 10, 100 or 200 mg/kg bw/day (reduced to 150 mg/kg bw/day on Day 31 for males and Day 30 for females) resulted in the unscheduled death of three males and two females in the high dose group. Following the further reduction of the high dose level to 75 mg/kg bw/day on Day 36 for males and Day 35 for females, no adverse effects were evident. These sporadic deaths were not associated with target organ histopathological changes which could have suggested that mortality or morbidity was associated with, or a consequence of, systemic toxicity. The evidence of degenerative and inflammatory changes in the upper respiratory tract of these animals supports the conclusion that these deaths were associated with the process of gavage dose administration of an irritant material and the pathology was due to gastric reflux.
The death of one male treated with 100 mg/kg bw/day was undetermined but was considered unrelated to treatment. There were no associated treatment related changes in the surviving 100 mg/kg bw/day animals, therefore the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be at least 100 mg/kg bw/day.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-08 to 1989-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
purity, storage conditions not reported
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: responsibility of the Sponsor

OTHER SPECIFICS:
- Name of test material (as cited in study report): 6398-10-1
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, Massachusetts
- Age at study initiation: 47 days old
- Weight at study initiation: approximately 135 to 215 g
- Fasting period before study: no
- Housing: vertical cage positioning in stainless steel 1/2" wire mesh cages. Cage sizes were in accordance w i t h the "Guide for the Care and Use of Laboratory Animals" of the Institute of Laboratory Animal Resources, National Research Council (NIH 86-23, 1985)
- Diet (e.g. ad libitum): Purina Certified Rodent Lab Meal , ad libitum
- Water (e.g. ad libitum): fresh tap water, ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C±3°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12h dark/12h light
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: clipped dorsal area
- Type of wrap if used: a gauze patch (2x2 cm) wrapped with an elastic bandage which was taped to the animal
- Time intervals for shavings or clipplings: weekly or when needed

REMOVAL OF TEST SUBSTANCE: no washing

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 5 ml/kg
- Constant volume or concentration used: yes

VEHICLE: deionized water

USE OF RESTRAINERS FOR PREVENTING INGESTION: no data
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
approximately 6 hours
Frequency of treatment:
once daily, five days per week for a period of 90 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
16 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
mid dose group
Dose / conc.:
160 mg/kg bw/day (nominal)
Remarks:
high dose group
No. of animals per sex per dose:
- Group I - 5 males + 5 females: control recovery group
- Groups II, III, and IV - 15 males + 15 females: low , mid and high dose groups
- Groups V and VI - 10 males + 10 females: control-recovery and high recovery dose groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based upon a 28 Day Dermal Toxicity Study in Rats:
The test substance was administered in a solution by dermal application to five groups of 10 rats (five males and five females per group) for 6 hours/day, 5 days/week for 28 days. The dose groups were 10, 50, 100, 250, and 500 mg/kg. An additional group of 10 rats (five male, five female) received the vehicle (deionized water) and served as a vehicle control group. No clinical signs were observed in the vehicle control group or the 10 and 50 mg/kg dose groups.
Clinical signs observed in the 100, 250 and 500 mg/kg treatment groups included very slight to severe erythema and extreme sloughing of skin, fissuring of skin, 15% to 75% necrosis of dose area, and scattered necrosis of dose area. The severity of these observations was dose dependent. Three rats died on study (one female in the high dose group on Day15 and two females in the high dose group on Day 17). Necropsy of the animals dying on study revealed mottled lungs and/or pale and prominent lobular patter of the liver. There were no statistically significant differences found in the group mean body weights, weight gains or daily food consumption. Terminal necropsy revealed mottled or pale lungs, red foci throughout lungs, tan foci and yellow-brown discoloration of the medial lobe of the liver, adhered lobes of the liver and mottled kidneys in the treatment groups. Terminal necropsy of the vehicle control group revealed mottled lungs, red foci throughout lungs and/or mottled kidneys. Based up on the results of this dose-range finding study, the suggested doses for the 90 -day study in rats were 16, 50 and 160 mg/kg.

- A negative control recovery group and a high dose recovery group remained on test untreated for an additional 28 days to determine reversibility, persistence, or delayed occurrence of toxic effects.


Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked in table [No.?] were included. cage side observation included, but were not limited to, changes in skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and during treatment at day 25, day 88 and day 116
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to initiation of the study and at sacrifice period
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, overnight
- How many animals: 10 (5/sex)
- Parameters examined: hemoglobin, hematocrit, erythrocyte count, total and differential leucocyte counts, platelet count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to initiation of the study and at sacrifice period
- Animals fasted: Yes, overnight
- How many animals: 10 (5/sex)
- Parameters examined: calcium, phosphorus, chloride, sodium, potassium, fasting glucose, serum alanine aminotransferase, serum aspartate aminotransferase, gamma glutamyl transpeptidase, urea nitrogen, albumin, globulin, blood creatinine, total bilirubin, total serum protein measurements

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- on all animals.
- examination of the external surface of the body, all orifices, and the cranial, thoracic, abdaminal and pelvic cavities and their contents.
- the following organs were weighed: liver, gonads, kidneys, adrenal glands, brain

HISTOPATHOLOGY: Yes
- the following organs and tissues were fixed and preserved for histopathological examination: gross lesions, brain-including sections of medulla/pons, cerebellar cortex and cerebral cortex, pituitary, thyroid/parathyroid, thymus, lungs (intact), trachea, heart, sternum with bone marrow, salivary glands, liver, spleen, kidneys/adrenals, pancreas, gonads, uterus, accessory genital organs (epididymides, prostate, and if present, seminal vesicles), aorta, skin (treated and uncreaced areas), esophagus, nasal turbinates, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, representative lymph node (submandibular), mammary gland, thigh musculature, peripheral nerve, eyes, femur-including arricular surface, spinal cord at three levels - cervical, midthoracic and lumbar, exorbital lachrymal glands
Statistics:
Evaluation of equality of means was made by the one way analysis of variance using the F distribution to assess significance. If significant differences among the means were indicated, Dunnett's or Student's t-test was used to determine significant differences from control means.
Clinical signs:
no effects observed
Description (incidence and severity):
- no clinical signs of systematic toxicity observed during the study
- additional clinical observation: noted during the study and considered not to be related to test article
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
- test article related moderate to severe skin irritation was observed in the mid and high dose groups. The skin irritation was reversible after discontinuation of treatment during the recovery period
- control or low dose group for either sex: no evidence of dermal irritation; in mid dose males and females at day 33 and day 34 respectively: dermal irritation observed; in high dose males and females: day 2 and day 5 dermal irritation observed;
- incidence and severity of dermal irritation increased with both the dose and duration of treatment and included the following observation for both mid and high dose animals: erythema, edema, sloughing of skin, scattered areas of necrosis, 25% of the area necrotic, and alopecia
- high dose recovery males and females: dermal irritation of similar incidence and severity occured during the 90 day treatment period, however, there was no evidence of delayed toxicity for either sex during the reversibility period, except for very slight erythema in one of ten high dose recovery females dermal irritation was no longer evident after the 28 day reversibility period (Day 118)
Mortality:
no mortality observed
Description (incidence):
- 2 non-treatment related deaths occured during study (judged as coincidental)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- statistically significant decreases in bw detected: in the male high dose group on days 7, 14, 21 and 28 when compared to control values. After day 35 there was a dose-related decrease in bw in males through day 91; however, these differences were not statistically significant.
- no statistically significant differences observed in female body weights. No statistically significant differences were detected between recovery control and recovery high dose groups during the 90 day treatment or 28 day recovery period.
- a statistically significant decrease in the group mean body weight gains for high dose males on days 7 and 14 and for mid dose males on day 91 when compared to che control group.
- significant decreases in bw gains were observed for the male high recovery groups on days 7 and 56 and a significant increase on day 21 when compared to the recovery control group. A significant decrease was observed for the female high dose recovery group on day 7 and an increase on day 77 when compared to the recovery control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- a statistically significant decrease observed: in the group mean daily food consumption for the male high dose on Day 30. No other statistically significant differences were detected between non-recovery test article treated and control groups. Male high dose recovery group mean daily food
consumption was significantly increased on day 21 and day 84 when compared to the recovery control group. Female high dose recovery group mean daily food consumprion was significantly increased on days 91 and 118.
Food efficiency:
not specified
Description (incidence and severity):
No data
Water consumption and compound intake (if drinking water study):
not specified
Description (incidence and severity):
No data
Ophthalmological findings:
no effects observed
Description (incidence and severity):
- no ocular findings identified that were attributed to test article administration
- the lesions identified were consistent with sequela from a sialodacryoadenitis virus infection (striate retinopathy, keratitis and anterior synechia) or a congenial defect (disc coloboma)
- the lesions noted occurred in the following animals: On day 25, striate retinopathy was noted in Group II males (#9233-left eye; 9237 and 9239-right eye) and Group V males (#9321 and 9329-left eye); a disc coloboma was noted in a Group II male (#9239-left eye) and nasal keratitis occurred in Group II female (#9377-both eyes). On day 88, striate retinopathy was noted in a Group I female (#9224-left eye), a Group II male (#9239-right eye), a
Group IV male (#9300-right eye), two Group V males (#9329-left eye,; #9321-right eye) and a Group VI male (#9350-left eye). In addition, a group II male had anterior synechia (#9239-right eye) and a Group III female had keratitis (#9388-both eyes). On Day 118 a Group V male had striate retinopathy (#9329-left eye)
Haematological findings:
no effects observed
Description (incidence and severity):
- a few significant differences were noted in the hematological data (not dose dependent, within historical limits, not biologically significant or attributable to the administration of the test article)
- on day 30 there was a statistically significant decrease in male values for eosinophils (low and mid dose groups), lymphocytes, white blood cells (low, mid and high dose groups) and platelets (high dose group). There were no significant differences between control and test article treated females on day 30. On day 90, there w ere no significant differences between control and test article treated males. Statistically significant increases were noted for female hemoglobin and mean corpuscular hemoglobin concentration (high dose group) and red blood cell numbers (low, mid and high dose groups). On day 118, except for statistically significant decrease in platelets for high dose recovery females and a statistically significant decrease in eosinophils for high dose recovery males, there were no additional significant difference in the hematology values for recovery animals of either sex.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- several significant differences were noted in the clinical chemistry data
- evaluation of the clinical chemistry data just prior to the day 30 interim sacrifice revealed significantly lower male values for total protein (mid dose) and globulin (mid and high dose); lower female values for GGPT (low dose), total bilirubin (low and mid dose), blood urea nitrogen, AST and sodium (mid dose); and higher female values for calcium (mid dose).
- on Day 90, significantly lower male values for creatinine, total protein, albumin (low, mid and high dose), sodium and calcium (high dose) ; higher male chloride (low, mid and high dose), male phosphorus (mid and high dose) and GGPT (high dose). Significantly lower female values for blood urea nitrogen (mid and high dose) and creatinine (mid dose); higher female values for ALT, AST and sodium (low dose), phosphorus (low, mid and high dose) and potassium (high dose).
- clinical chemistry data evaluated just prior to the day 118 recovery necropsy revealed a significantly higher male potassium value for the high dose recovery males. There was significantly higher AST for the high dose recovery females
Urinalysis findings:
not specified
Description (incidence and severity):
No data
Behaviour (functional findings):
not specified
Description (incidence and severity):
No data
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- no statistically significant differences in absolute organ weights between the control and treated groups after 30 and 90 days of treatment or 28 days of recovery for either sex.
- a statistically significant decrease in fasted body weight was noted for the high dose males at the day 90 necropsy.
Gross pathological findings:
no effects observed
Description (incidence and severity):
- The majority of the gross findings at the day 30 (interim), day 90 (terminal) and day 118 (recovery) necropsies were non-specific and low incidence. Based upon histomorphologic evaluation of the selected tissues, there was no apparent relationship of these gross lesions to test article administration.
- At the day 30 necropsy, one high dose male, one mid dose female and two high dose females exhibited scattered or small scabs on the treatment site. At the day 90 necropsy, two high dose males exhibited multiple scabs and ulcers on treated skin and one high dose female exhibited multiple scabs on treated skin. At the day 118 (recovery) necropsy there were no gross lesions detected in the treated or untreated skin sites for either sex.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- except for the skin irritation at the treatment site of the mid and high dose , there were no histomorphological alterations attributable to the administration of the test article
Histopathological findings: neoplastic:
not specified
Description (incidence and severity):
No data
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
> 160 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: no clinical signs of systemic toxicity were attributable to the administration of the test article (160 mg/kg/d or less)
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Critical effects observed:
not specified
Conclusions:
Based upon the results of this subchronic 90 day dermal toxicity study in rats, the test substance did not produce systemic toxicity when administered dermally five days per week for 30 and 90 days at doses of 16, 50 and 160 mg/kg. Therefore, the NOAEL was established as greater than 160 mg/kg body weight/day.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
160 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-08 to 1989-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
purity, storage conditions not reported
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: responsibility of the Sponsor

OTHER SPECIFICS:
- Name of test material (as cited in study report): 6398-10-1
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, Massachusetts
- Age at study initiation: 47 days old
- Weight at study initiation: approximately 135 to 215 g
- Fasting period before study: no
- Housing: vertical cage positioning in stainless steel 1/2" wire mesh cages. Cage sizes were in accordance w i t h the "Guide for the Care and Use of Laboratory Animals" of the Institute of Laboratory Animal Resources, National Research Council (NIH 86-23, 1985)
- Diet (e.g. ad libitum): Purina Certified Rodent Lab Meal , ad libitum
- Water (e.g. ad libitum): fresh tap water, ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C±3°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12h dark/12h light
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: clipped dorsal area
- Type of wrap if used: a gauze patch (2x2 cm) wrapped with an elastic bandage which was taped to the animal
- Time intervals for shavings or clipplings: weekly or when needed

REMOVAL OF TEST SUBSTANCE: no washing

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 5 ml/kg
- Constant volume or concentration used: yes

VEHICLE: deionized water

USE OF RESTRAINERS FOR PREVENTING INGESTION: no data
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
approximately 6 hours
Frequency of treatment:
once daily, five days per week for a period of 90 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
16 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
mid dose group
Dose / conc.:
160 mg/kg bw/day (nominal)
Remarks:
high dose group
No. of animals per sex per dose:
- Group I - 5 males + 5 females: control recovery group
- Groups II, III, and IV - 15 males + 15 females: low , mid and high dose groups
- Groups V and VI - 10 males + 10 females: control-recovery and high recovery dose groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based upon a 28 Day Dermal Toxicity Study in Rats:
The test substance was administered in a solution by dermal application to five groups of 10 rats (five males and five females per group) for 6 hours/day, 5 days/week for 28 days. The dose groups were 10, 50, 100, 250, and 500 mg/kg. An additional group of 10 rats (five male, five female) received the vehicle (deionized water) and served as a vehicle control group. No clinical signs were observed in the vehicle control group or the 10 and 50 mg/kg dose groups.
Clinical signs observed in the 100, 250 and 500 mg/kg treatment groups included very slight to severe erythema and extreme sloughing of skin, fissuring of skin, 15% to 75% necrosis of dose area, and scattered necrosis of dose area. The severity of these observations was dose dependent. Three rats died on study (one female in the high dose group on Day15 and two females in the high dose group on Day 17). Necropsy of the animals dying on study revealed mottled lungs and/or pale and prominent lobular patter of the liver. There were no statistically significant differences found in the group mean body weights, weight gains or daily food consumption. Terminal necropsy revealed mottled or pale lungs, red foci throughout lungs, tan foci and yellow-brown discoloration of the medial lobe of the liver, adhered lobes of the liver and mottled kidneys in the treatment groups. Terminal necropsy of the vehicle control group revealed mottled lungs, red foci throughout lungs and/or mottled kidneys. Based up on the results of this dose-range finding study, the suggested doses for the 90 -day study in rats were 16, 50 and 160 mg/kg.

- A negative control recovery group and a high dose recovery group remained on test untreated for an additional 28 days to determine reversibility, persistence, or delayed occurrence of toxic effects.


Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked in table [No.?] were included. cage side observation included, but were not limited to, changes in skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and during treatment at day 25, day 88 and day 116
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to initiation of the study and at sacrifice period
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, overnight
- How many animals: 10 (5/sex)
- Parameters examined: hemoglobin, hematocrit, erythrocyte count, total and differential leucocyte counts, platelet count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to initiation of the study and at sacrifice period
- Animals fasted: Yes, overnight
- How many animals: 10 (5/sex)
- Parameters examined: calcium, phosphorus, chloride, sodium, potassium, fasting glucose, serum alanine aminotransferase, serum aspartate aminotransferase, gamma glutamyl transpeptidase, urea nitrogen, albumin, globulin, blood creatinine, total bilirubin, total serum protein measurements

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- on all animals.
- examination of the external surface of the body, all orifices, and the cranial, thoracic, abdaminal and pelvic cavities and their contents.
- the following organs were weighed: liver, gonads, kidneys, adrenal glands, brain

HISTOPATHOLOGY: Yes
- the following organs and tissues were fixed and preserved for histopathological examination: gross lesions, brain-including sections of medulla/pons, cerebellar cortex and cerebral cortex, pituitary, thyroid/parathyroid, thymus, lungs (intact), trachea, heart, sternum with bone marrow, salivary glands, liver, spleen, kidneys/adrenals, pancreas, gonads, uterus, accessory genital organs (epididymides, prostate, and if present, seminal vesicles), aorta, skin (treated and uncreaced areas), esophagus, nasal turbinates, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, representative lymph node (submandibular), mammary gland, thigh musculature, peripheral nerve, eyes, femur-including arricular surface, spinal cord at three levels - cervical, midthoracic and lumbar, exorbital lachrymal glands
Statistics:
Evaluation of equality of means was made by the one way analysis of variance using the F distribution to assess significance. If significant differences among the means were indicated, Dunnett's or Student's t-test was used to determine significant differences from control means.
Clinical signs:
no effects observed
Description (incidence and severity):
- no clinical signs of systematic toxicity observed during the study
- additional clinical observation: noted during the study and considered not to be related to test article
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
- test article related moderate to severe skin irritation was observed in the mid and high dose groups. The skin irritation was reversible after discontinuation of treatment during the recovery period
- control or low dose group for either sex: no evidence of dermal irritation; in mid dose males and females at day 33 and day 34 respectively: dermal irritation observed; in high dose males and females: day 2 and day 5 dermal irritation observed;
- incidence and severity of dermal irritation increased with both the dose and duration of treatment and included the following observation for both mid and high dose animals: erythema, edema, sloughing of skin, scattered areas of necrosis, 25% of the area necrotic, and alopecia
- high dose recovery males and females: dermal irritation of similar incidence and severity occured during the 90 day treatment period, however, there was no evidence of delayed toxicity for either sex during the reversibility period, except for very slight erythema in one of ten high dose recovery females dermal irritation was no longer evident after the 28 day reversibility period (Day 118)
Mortality:
no mortality observed
Description (incidence):
- 2 non-treatment related deaths occured during study (judged as coincidental)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- statistically significant decreases in bw detected: in the male high dose group on days 7, 14, 21 and 28 when compared to control values. After day 35 there was a dose-related decrease in bw in males through day 91; however, these differences were not statistically significant.
- no statistically significant differences observed in female body weights. No statistically significant differences were detected between recovery control and recovery high dose groups during the 90 day treatment or 28 day recovery period.
- a statistically significant decrease in the group mean body weight gains for high dose males on days 7 and 14 and for mid dose males on day 91 when compared to che control group.
- significant decreases in bw gains were observed for the male high recovery groups on days 7 and 56 and a significant increase on day 21 when compared to the recovery control group. A significant decrease was observed for the female high dose recovery group on day 7 and an increase on day 77 when compared to the recovery control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- a statistically significant decrease observed: in the group mean daily food consumption for the male high dose on Day 30. No other statistically significant differences were detected between non-recovery test article treated and control groups. Male high dose recovery group mean daily food
consumption was significantly increased on day 21 and day 84 when compared to the recovery control group. Female high dose recovery group mean daily food consumprion was significantly increased on days 91 and 118.
Food efficiency:
not specified
Description (incidence and severity):
No data
Water consumption and compound intake (if drinking water study):
not specified
Description (incidence and severity):
No data
Ophthalmological findings:
no effects observed
Description (incidence and severity):
- no ocular findings identified that were attributed to test article administration
- the lesions identified were consistent with sequela from a sialodacryoadenitis virus infection (striate retinopathy, keratitis and anterior synechia) or a congenial defect (disc coloboma)
- the lesions noted occurred in the following animals: On day 25, striate retinopathy was noted in Group II males (#9233-left eye; 9237 and 9239-right eye) and Group V males (#9321 and 9329-left eye); a disc coloboma was noted in a Group II male (#9239-left eye) and nasal keratitis occurred in Group II female (#9377-both eyes). On day 88, striate retinopathy was noted in a Group I female (#9224-left eye), a Group II male (#9239-right eye), a
Group IV male (#9300-right eye), two Group V males (#9329-left eye,; #9321-right eye) and a Group VI male (#9350-left eye). In addition, a group II male had anterior synechia (#9239-right eye) and a Group III female had keratitis (#9388-both eyes). On Day 118 a Group V male had striate retinopathy (#9329-left eye)
Haematological findings:
no effects observed
Description (incidence and severity):
- a few significant differences were noted in the hematological data (not dose dependent, within historical limits, not biologically significant or attributable to the administration of the test article)
- on day 30 there was a statistically significant decrease in male values for eosinophils (low and mid dose groups), lymphocytes, white blood cells (low, mid and high dose groups) and platelets (high dose group). There were no significant differences between control and test article treated females on day 30. On day 90, there w ere no significant differences between control and test article treated males. Statistically significant increases were noted for female hemoglobin and mean corpuscular hemoglobin concentration (high dose group) and red blood cell numbers (low, mid and high dose groups). On day 118, except for statistically significant decrease in platelets for high dose recovery females and a statistically significant decrease in eosinophils for high dose recovery males, there were no additional significant difference in the hematology values for recovery animals of either sex.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- several significant differences were noted in the clinical chemistry data
- evaluation of the clinical chemistry data just prior to the day 30 interim sacrifice revealed significantly lower male values for total protein (mid dose) and globulin (mid and high dose); lower female values for GGPT (low dose), total bilirubin (low and mid dose), blood urea nitrogen, AST and sodium (mid dose); and higher female values for calcium (mid dose).
- on Day 90, significantly lower male values for creatinine, total protein, albumin (low, mid and high dose), sodium and calcium (high dose) ; higher male chloride (low, mid and high dose), male phosphorus (mid and high dose) and GGPT (high dose). Significantly lower female values for blood urea nitrogen (mid and high dose) and creatinine (mid dose); higher female values for ALT, AST and sodium (low dose), phosphorus (low, mid and high dose) and potassium (high dose).
- clinical chemistry data evaluated just prior to the day 118 recovery necropsy revealed a significantly higher male potassium value for the high dose recovery males. There was significantly higher AST for the high dose recovery females
Urinalysis findings:
not specified
Description (incidence and severity):
No data
Behaviour (functional findings):
not specified
Description (incidence and severity):
No data
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- no statistically significant differences in absolute organ weights between the control and treated groups after 30 and 90 days of treatment or 28 days of recovery for either sex.
- a statistically significant decrease in fasted body weight was noted for the high dose males at the day 90 necropsy.
Gross pathological findings:
no effects observed
Description (incidence and severity):
- The majority of the gross findings at the day 30 (interim), day 90 (terminal) and day 118 (recovery) necropsies were non-specific and low incidence. Based upon histomorphologic evaluation of the selected tissues, there was no apparent relationship of these gross lesions to test article administration.
- At the day 30 necropsy, one high dose male, one mid dose female and two high dose females exhibited scattered or small scabs on the treatment site. At the day 90 necropsy, two high dose males exhibited multiple scabs and ulcers on treated skin and one high dose female exhibited multiple scabs on treated skin. At the day 118 (recovery) necropsy there were no gross lesions detected in the treated or untreated skin sites for either sex.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- except for the skin irritation at the treatment site of the mid and high dose , there were no histomorphological alterations attributable to the administration of the test article
Histopathological findings: neoplastic:
not specified
Description (incidence and severity):
No data
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
> 160 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: no clinical signs of systemic toxicity were attributable to the administration of the test article (160 mg/kg/d or less)
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Critical effects observed:
not specified
Conclusions:
Based upon the results of this subchronic 90 day dermal toxicity study in rats, the test substance did not produce systemic toxicity when administered dermally five days per week for 30 and 90 days at doses of 16, 50 and 160 mg/kg. Therefore, the NOAEL was established as greater than 160 mg/kg body weight/day.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

Repeated dose toxicity: dermal

Based upon the results of the subchronic 90-day dermal repeated dose toxicity study in rats, performed by Pharmakon Research International, Inc (1990) according to the OECD guideline 411, moderate to severe skin irritation was observed in the mid (50 mg/kg bw/d) and high (160 mg/kg bw/d) dose groups. The skin irritation was reversible after discontinuation of treatment during the recovery period. In addition, adverse effects were observed for body weight, food consumption, clinical chemistry and organ weights. These differences (except skin irritation) were within historical limits and are considered not biologically significant or attributable to the administration of the test article. In conclusion, the test substance did not produce systemic toxicity when administered dermally five days per week for 30 and 90 days at doses of 16, 50 and 160 mg/kg. Therefore, it is concluded that the NOAEL for systemic effects is above 160 mg/kg bw/day.

Repeated dose toxicity: oral

The oral (gavage) administration of the test substance for up to ninety consecutive days, to Wistar rats of both sexes at dose levels of 10, 100 or 200 mg/kg bw/day (reduced to 150 mg/kg bw/day on Day 31 for males and Day 30 for females and then further reduced to 75 mg/kg bw/day on Day 36 for males and Day 35 for females) resulted in the early death of three males and two females in the high dose group and the early termination of one male treated with 100 mg/kg bw/day. 

The two females treated with 200 mg/kg bw/day that were found dead and one of the males from this treatment group that was found dead on Day 16 did not show any clinical signs prior to death. The male that was found dead on Day 31 showed labored respiration, decreased respiratory rate, prostration, and clonic convulsions on Day 30. The male that was found dead on Day 35, only previously showed increased salivation and the male treated with 100 mg/kg bw/day that was killed in extremis on Day 83 showed noisy/gasping respiration, decreased respiratory rate, prostration and clonic convulsions. At necropsy, one of the high dose females that was found dead on Day 23 did not show any macroscopic abnormalities. The other high dose female that was found dead on Day 23 had an enlarged stomach with a thin glandular and non-glandular region, gaseous distension in the stomach, cecum and colon, a dark liver, thymus and uterus and a green discolored mammary gland. The high dose male that was found dead on Day 16 had a dark liver, small seminal vesicles, a thin non-glandular region of the stomach and a hole in the esophagus. The other two high dose males that were found dead had reddened lungs and the male which died on day 35 also had a dark liver. The male treated with 100 mg/kg bw/day that was killed in extremis on day 83 had a raised limiting ridge in the stomach and a reddened glandular region of the stomach. Histopathological examination of the premature decedents at the high dose group revealed regenerative hyperplasia in the trachea, ulceration in the nasopharynx and/or inflammatory changes in the nasopharynx in four animals. Minimal increased hematopoiesis in the spleen was also evident in one of these males and one male also had a tear in the esophagus caused by a dosing accident. The cause of death for three of these animals was considered likely to have been reflux and in the fourth, the hole in the esophagus. In the remaining female that was found dead there was notable autolysis, however, the cause of death was undetermined. The male treated with 100 mg/kg bw/day that was killed in extremis on Day 83 had no notable changes and the cause of death was undetermined.

For all sporadic deaths there were no associated target organ histopathological changes to suggest that mortality or morbidity was associated with, or a consequence of, systemic toxicity. The evidence of degenerative and inflammatory changes in the upper respiratory tract of these animals supports the conclusion that these deaths were associated with the process of gavage dose administration of this material; the aetiology of the pathology is considered to be gastric reflux. It is recognised that rats do not demonstrate the normal emetic reflex that is seen in humans, however, the mechanical process of intubation of the oesophagus/stomach with a catheter and subsequent introduction of the test item into the stomach may induce a minor reflux of gastric contents at the time of withdrawal of the catheter. This process of gastric reflux has been reported by Damsch et al as an observation in other studies. The authors reported that physical changes to structures such as the upper respiratory tract can take place as a result of gastric reflux. These changes may be sufficient to result in mortality. Respiratory changes prior to death were the most common findings observed for this study. The pathological changes may extend into the nasal structures which indicate the extent of pathological change that may result from gastric reflux. In a separate study in rats where chronic acid reflux oesophagitis was surgically induced, inflammatory changes in the larynx and pharynx could be identified. The clinical effects of gastric/oesophageal reflux have been associated with respiratory disease in humans. Human ingestion of strong irritants such as ammonia can result in aspiration of the material resulting in a condition described as acute respiratory distress syndrome. The animals that died on this study generally showed minimal clinical signs of ill-health which suggests that effects leading to mortality were spontaneous and not necessarily related to cumulative toxic effects. This is supported by a lack of consistency with the timing of mortalities on the study. The conclusions that may therefore be drawn from the mortalities on this study are as follows:

       i.           A combination of factors resulted in the mortalities. The primary cause was considered to be the process of orally intubating a potentially irritant material to rats. This physical process is likely to have resulted in partial reflux of gastric contents, affecting the upper respiratory tract.

     ii.           It is likely that the extent of gastric reflux determined the clinical effects observed. In its mildest form there was evidence of post dosing salivation, which progressed to animals with noisy respiration and ultimately animals with severe respiratory distress and morbidity/mortality.

   iii.           The appearance of the findings was not predictable but was more prevalent at higher concentrations of the test item. The time to appearance of reactions to treatment suggests the animals were demonstrating progressive reactions to the dose administration process.

   iv.           The lack of clinical effects and subsequent pathology evaluations clearly confirms that there was no underlying systemic toxicity which could have prompted such mortalities.

Increased salivation and noisy respiration were also evident in the surviving animals of both sexes treated at the high dose level and both sexes treated with 100 mg/kg bw/day. In addition, a low incidence of noisy respiration at 10 mg/kg bw/day may also be a consequence of the dose administration process. Body weight gain was initially affected at the high dose level in either sex, however, following the reduction of the dose level, improvement was evident thereafter. Food consumption for all treated animals was unaffected.

Hematological investigations revealed changes in white blood cell counts in either sex treated with the high dose level only. Although the majority of individual values were above historical control ranges, no associated histopathological changes were evident at the end of the treatment period. Therefore, these changes may be the delayed result of the initial insult by the test item when the animals were treated with 200 mg/kg bw/day and as no further toxicity was evident at 75 mg/kg bw/day, these changes can be regarded as non-adverse. 

The clinical signs evident in the male treated with 100 mg/kg bw/day that was killed in extremis were the same observations as those seen in one of the premature decedents at the high dose level. Whilst there were no histopathological changes to the upper respiratory tract for this animal the observation of significant respiratory changes prior to termination do lead to the conclusion that morbidity was due to the same cause as that seen amongst decedents at the higher dose initial dose level of 200/150 mg/kg bw/day. However, based on the lack of any significant systemic toxicity observed amongst animals at 100 mg/kg bw/day as observed by the lack of any significant histopathology or blood chemistry/haematology changes plus no significant effects on body weight development or food consumption the appropriate interpretation of a No Observed Adverse Effect Level would be 100 mg/kg bw /day or above. A higher dose level could not be used for this as the number of mortalities and the initial effects upon body weight development at a dose level of 200 mg/kg bw/day would suggest that this dose concentration could not clearly be justified as a suitable No Adverse Effect Level.

 

Repeated dose toxicity: inhalation

No reliable data were available for this exposure route. This type of study does not need to be performed, since a 90 -day repeated dose toxicity study is available via dermal and via oral route of administration (REACH Regulation, Annex IX, Column 2 adaptation). Moreover, the inhalation route is not expected to be the main route of exposure.

A detailed discussion on the repeated dose toxicity study and its data interpretation is included in section 13.

Justification for classification or non-classification

Based on the results of the repeated dose toxicity study via the dermal and oral route, the substance should not be classified for specific target organ toxicity - repeated exposure.