1,1'-[[3-(dimethylamino)propyl]imino]bispropan-2-ol

Endpoint summary

Description of key information

Repeated dose toxicity - oral : Two key studies are available. Martell (2013) performed a combined repeated dose toxicity study with reproduction/developmental toxicity screening test in rats according to OECD guideline 422

and according to GLP requirements. A NOAEL value > 500 mg/kg bw/day was derived for male systemic and maternal systemic toxicity. Malleshappa (2021) performed a 90-day oral repeated dose toxicity study in rats (K1) according to the OECD guideline 408 and according to GLP requirements.

A NOAEL value for systemic toxicity of 150 mg/kg/day was derived.

 

Repeated dose toxicity - dermal : No repeated dose toxicity via dermal administration is required.

 

Repeated dose toxicity - inhalation : No repeated dose toxicity via inhalatory administration is required.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-12-28 to 2021-07-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 25, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): JEFFCAT® DPA catalyst
- lot/batch number: PFW190123
- Physical appearance: Liquid
- Manufactured date: 04.03.2019
- Retest date: 03.06.2022
- pH: 11,7
- Purity: This product is not specified by purity/assay but total titratable amine content. Amine content: 9.0 meq/g. Typical purity: 88.74 A%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (+15 to +25ºC) Store under inert atmosphere, at temperature no higher than 22ºC.
- Stability and homogeneity of the test material in the vehicle under test conditions and during storage: The stability of the test item in the vehicle was established at 1 and 100 mg/mL under Eurofins Advinus Study No. G21131. Based on the interim results, the test item was stable in the vehicle up to to 24 hours when stored at room temperature and 5 days at refrigerated condition.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: not specified
- Reactivity of the test material with the incubation material used (e.g. plastic ware): not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): not specified

FORM AS APPLIED IN THE TEST: liquid

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: 40 male and 40 female Wistar rats, Hylasco Biotechnology (India)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at start of treatment: 6-7 weeks
- Weight at start of treatment: Males : 0.195 to 0.246 kg, Females: 0.153 to 0.192 kg
- Fasting period before study: not specified
- Housing: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes. Polycarbonate rat huts were provided to the animals as environmental enrichment objects and changed along with cage at least once a week. During the experimental period, animals were housed in a single experimental room of barrier area (SC-15). Steam sterilized corn cob was used as bedding and changed along with the cage at least twice a week.
- Diet: ad libitum, Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany
- Water: ad libitum, Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd. Mumbai 400 001, India, was provided to rats in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: five days before start of the treatment

DETAILS OF FOOD AND WATER QUALITY: The feed and water provided to the rats was tested for contaminants. Based on the latest analytical certificate/s available, there were no known contaminants in the food, water and bedding that are expected to interfere with the results of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 24°C
- Humidity (%): 49 to 66 %
- Air changes (per hr): 12.5-14.3 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2021-01-05 To: 2021-05-05

Route of administration:

oral: gavage

Details on route of administration:

The dose formulations were administered orally by gavage to specific group of rats once daily at approximately the same time (+/-3 hours) each day for 90 consecutive days.

Vehicle:

water

Remarks:

Milli-Q water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- pre-calibration of the beaker to desired volume: Milli-Q® water was measured in a graduated cylinder to the final volume of 60 mL. The measured water was transferred into a clean beaker and upper and lower meniscus of water was marked on the beaker using a marker. The water was discarded and the beaker was dried.
- Required quantities of the test item was weighed in a beaker (previously calibrated to a desired volume*) for each dose levels separately and a small volume of vehicle (Milli-Q® water) was added and stirred using a glass rod till a uniform solution was obtained. The volume was made up to the mark using the vehicle to get the final desired concentration of 7.5, 15 and 45 mg/mL for the G2, G3 and G4 groups, respectively. The solutions were mixed well by stirring using a magnetic stirrer.
- the dose formulations were prepared once daily before start of each day of dosing
- the volume of dose formulation prepared was varied depending on the requirement and/or body weights of the rats during experimental period.

VEHICLE
- Concentration in vehicle: 7.5, 15 and 45 mg/mL for the G2, G3 and G4 groups, respectively.
- Amount of vehicle (if gavage): 10 mL/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For test item concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during 2nd (Day 35) and 3rd (Day 71) month of the treatment period and analysed in-house. For each set, composite sample was drawn from each preparation and in case of control, duplicate composite sample was drawn.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G21131. One set of samples were analyzed and other set (second set) of samples were stored at room temperature for possible reanalysis as a backup and these samples were discarded, as the analysis results of first set of samples were within the acceptable limits.
Formulations were considered acceptable as overall mean results were within ± 10.0 % of the claimed concentration and relative standard deviation (% RSD) was less than 10.0 %.

Duration of treatment / exposure:

90 consecutive days

Frequency of treatment:

Daily at approximately the same time (± 3 hours)

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

G1, vehicle control

Dose / conc.:

75 mg/kg bw (total dose)

Remarks:

G2, Low dose

Dose / conc.:

150 mg/kg bw (total dose)

Remarks:

G3, Mid dose

Dose / conc.:

450 mg/kg bw (total dose)

Remarks:

G4, High dose

No. of animals per sex per dose:

Each group in the experiment was comprised of ten male and ten female rats.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels of 75 (G2), 150 (G3) and 450 (G4) mg/kg/day were selected for this study based on the results of an available OECD 422 study conducted by sponsor where the NOAEL was considered to be > 500 mg/kg bw/day (nominal dose received) for male and systemic toxicity and maternal systemic toxicity, and greater than 500 mg/kg bw/day for embryo-fetal toxicity in consultation with the Sponsor. In addition to the test doses, vehicle control group were included.

- Route of administration and justification: Route of test item administration was through oral gavage. The oral route was chosen because it provides an exaggerated model of possible exposure in humans.

- Justification for Selection of Vehicle: The Formulation Analytical Method Validation and Stability was established in Milli-Q water under Eurofins Advinus study (G21131). Hence, Milli-Q water was used as vehicle for dose formulation preparation.

Observations and examinations performed and frequency:

MORBIDITY and MORTALITY: Yes
- all rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon except during holidays wherein the observation was done once daily as there were no clinical signs observed.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: each rat was observed for checking general clinical signs twice daily during treatment period. On the days of scheduled detailed clinical examination, clinical signs were included as a part of detailed clinical observations except on day 1 wherein detailed clinical examination was done prior to the treatment and observations for general clinical signs was done after dosing the animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examination was done prior to the test item administration on Day 1 and at weekly intervals thereafter (± 1 days) during treatment period.
- During detailed clinical examination, all rats were observed cage-inside/outside for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size,
unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour (e.g. selfmutilation, walking backwards).

FUNCTIONAL OBSERVATION BATTERY TESTS
- Time schedule: performed during the 13th week (day 85) of treatment period
- home cage observations: Each rat was observed in the home cage for posture and for presence or absence of abnormal vocalizations, tremors and convulsions
- observations during removal of animal from home cage and handling: The objective of this phase of neurological examination was to observe the subject’s response to handling and to conduct other procedures of the FOB that can best be performed when the rat is being held. Each rat was observed for the following examinations: ease of removal from home cage, handling reactivity,
palpebral closure, eye examination, piloerection, lacrimation, salivation, skin/fur examination, perineum wetness, respiration, muscle tone and extensor thrust response. The observations were recorded using scores/ranks.
- open field observation: Rat was placed (one at a time) in an open arena, on a flat surface with a clean absorbent paper and observed for at least 2 minutes. Absorbent paper was replaced for each group. During this observation period, rat was evaluated as it moves about freely/unperturbed and the following observations were made and observations were recorded using core/ranks: gait, posture, tremors, mobility score, arousal level, clonic or tonic movements, stereotypic behaviour, bizarre behaviour, urination, defecation, rearing, abnormal vocalizations
- functional tests: Functional testing includes motor activity, sensory evaluation, landing hindlimbs footsplay and measurement of grip performance. The Opto-Varimex 5 data was analyzed in 10 minutes intervals and the same was reported
- sensory reactivity measurements: After the 2 minutes (approximately) observation period, while the rat was in the open field arena, the following tests were conducted. The rat was allowed to move freely in the open field box for these tests but positioned in the box by the observer in order to administer stimulus. During sensory reactivity measurements, rats were observed for following and the observations were recorded using scores/ranks. Observations: approach response, touch response, click response, tail-pinch response, pupil response, aerial righting reflex,
- landing hindlimbs footsplay: The landing hind limbs foot splay was performed by dropping the rat onto a
horizontal surface of the tabletop from a short height and measuring the distance between the hind feet upon landing. The hind feet of the rat were gently pressed to an ink pad just prior to testing. The rat was suspended in a prone position and then dropped from a height of approximately 30 cm on to a SOP format, which contains the details such as Study no., Animal no, Group and Sex. A clean recording SOP format was used for each rat. A total of 3 readings were recorded for each rat and average of 3 footsplay values is presented in the report along with the individual footsplay values.
- grip performance: Hindlimbs and forelimbs grip performance was tested using computerized dual
grip strength meter (Model: Columbus Instruments). Three trials were conducted for each rat i.e., three trials each for forelimb and hind limbs. Averages of three trials for both forelimb and hindlimbs are calculated and presented in the report along with the individual grip strength values
- physiological observations: Body temperature (rectal temperature) was measured in degree Celsius (°C) using digital thermometer. At the end of the functional test, body weight of each rat was measured

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights (g) were recorded prior to test item administration on Day 1 and weekly thereafter (± 1 day) for all groups of rats during treatment period. Fasting body weight was recorded prior to sacrifice for all surviving toxicity group animals.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The food consumption was measured at weekly intervals (± 1 day) during treatment period. The cage wise average food consumption (g/rat/day) was calculated


WATER CONSUMPTION AND COMPOUND INTAKE : No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examination of all animals was performed with an ophthalmoscope prior to start of the treatment and at the end of the treatment period. Before examination, mydriasis was induced using a 1 % solution of Tropicamide.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, approximately 4.0 mL of blood was collected.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, fasted overnight (water allowed)
- blood was collected with a fine capillary tube, by retro-orbital sinus puncture (anti-coagulant: K2 EDTA for hematology; tridosium citrate for coagulation)
- How many animals: 40
- parameters: red blood corpuscles, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, reticulocytes count, white blood corpuscles, differential leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), platelets, red blood cell morphology (red cell distribution width, haemoglobin distribution width, hyperchromic cells, hypochromic cells, macrocytes, microcytes, RBC fragments, RBC ghosts)
- Coagulation: Blood samples collected for coagulation analysis were centrifuged at 2500 times gravity (xg) for 10 minutes 15ºC for separation of plasma and analysed for the following parameters in plasma sample using STart Max coagulation analyzer (Diagnostica stago, 92600 Asnieres, France)
- parameters: prothrombin time, activated partial thromboplastin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, approximately 4.0 mL of blood was collected.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes , fasted overnight (water allowed)
- blood was collected with a fine capillary tube, by retro-orbital sinus puncture (anti-coagulant: lithium heparin)
- How many animals: 40
- Parameters: alanine aminotransferase, alkaline phosphatase, albumin, albumin/globulin ratio (calculated value), aspartate aminotransferase, blood urea nitrogen, chloride, creatinine, calcium, glucose, globulin (calculated value), HDL cholesterol, inorganic phosphorus, LDL cholesterol, potassium, sodium, total cholesterol, total plasma protein, triglycerides, total bilirubin

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: At the end of the treatment period, approximately 4.0 mL of blood was collected.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes , fasted overnight (water allowed)
- blood was collected with a fine capillary tube, by retro-orbital sinus puncture
- How many animals: 40
- blood samples collected in plain labeled tubes were kept on bench top for approximately 60 minutes before centrifugation. Serum was separated by centrifuging the whole blood samples at 5000 rpm for 5 minutes at 5 °C. The serum samples were placed in labeled plastic tubes and stored at +/- -70 °C until they were analyzed. Thyroid hormones were estimated by ELISA using Bio-RAD microplate washer and BIO-RAD model 680 readers.
- Hormones estimated: rodent thyroid stimulating hormone (TSH), rodent thyroxine (T4), rodent triiodothyronine (T3)

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected from all rats at the end of the treatment period in the urine collection tube.
- Metabolism cages used for collection of urine: Yes, each rat was placed overnight in a specially fabricated cage (water allowed) and the next morning the collected urine was sent for analysis.
- Animals fasted: yes, water allowed
- Parameters: specific gravity, nitrite, pH, proteins, glucose, ketone bodies, urobilinogen, bilirubin, appearance (colour and clarity), volume (approximate)
- microscopic examination: crystals, epithelial cells, casts

NEUROBEHAVIOURAL EXAMINATION: Yes
- functional battery tested, as described above

IMMUNOLOGY: No

OTHER:
- Oestrous Cycle Evaluation: Vaginal smear was examined in the female rats and the stage of oestrous cycle was recorded prior to necropsy.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All rats at the end of treatment period were subjected to detailed necropsy and findings were recorded. The necropsy included examination of external surfaces of the body, all orifices, cranial, thoracic and abdominal cavities and their contents). Terminal body weights were recorded for all animals immediately
prior to sacrifice. All rats were fasted overnight (water allowed), euthanized with isoflurane anesthesia, exsanguinated and subjected for gross examination.
- tissue collection, weighing and preservation: On completion of gross pathology examination, the tissues and organs were collected and weighed from all rats. (see table in 'additional information'-field . The tissues were preserved in 10% Neutral Buffered Formalin (NBF) except for the testes
and eyes.

ORGAN WEIGHTS:
- The organ weight ratios (organ to body weight and organ to brain weight) as percentage of fasting body weight and brain weight were determined and presented in the report. The paired organs were weighed together, and combined weight was presented.

HISTOPATHOLOGY: Yes
- Histopathological examination was carried out on the preserved organs of the vehicle control (G1) and high dose group animals (G4).
- Histopathological examination of the testes included a qualitative assessment of stages of
spermatogenesis.
- all gross lesions from all the animals were examined microscopically. Liver, lungs, spleen and mandibular lymph nodes were examined in lower dose (G2 and G3) group rats as test item-related changes were noted in these organs at high dose (G4).
- The tissues were processed for routine paraffin embedding and 4-5-micron sections were stained with Haematoxylin and Eosin stain. In addition, testes were sectioned at 3-4 μm and stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis. Unused tissues were archived.

Statistics:

Data was captured using the ProvantisTM laboratory information management system (LIMS). Parameters such as body weight, body weight change, body temperature, hindlimbs footsplay, grip performance, food consumption, organ weights, organ weight ratios (organ to body weight and organ to brain weight), laboratory Investigations – Haematology, Coagulation & Clinical Chemistry and transferred (motor activity, thyroid profile) data was evaluated using the Levene
Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA), when data found to be nonhomogeneous or of non-normal distribution, the data was subjected for transformation and ANOVA was performed on transformed data. When ANOVA found to be significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.
Data captured outside of ProvantisTM: The statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Ver.12.0. Descriptive statistics Mean, SD, Percentages & Numbers were presented by Treatment group and Day.
All hypothesis testing was carried out at the 5% (2-sided) significance level unless otherwise specified. Significant differences were designated throughout the report as below:
*: Statistically significant difference from the control group at p < 0.05

Clinical signs:

no effects observed

Description (incidence and severity):

At 450 mg/kg/day, transient clinical sign of slight salivation was observed soon after the dose administration in all animals from treatment Day 21. Nevertheless, the symptom subsided within a few minutes and the rats were found to be normal.
There were no clinical signs or mortalities observed during the treatment at 75 and 150 mg/kg bwt/day dose group in either sex.

Mortality:

no mortality observed

Description (incidence):

There were no mortalities observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment did not affect the mean body weights in all the tested doses in either sex during the treatment period.
Significantly lower absolute body weight gain during days 8-15 at all the tested doses, during days 79-85 at mid and high doses and significantly higher absolute weight gain during days 64-71 in mid and high dose males was observed. In females, significantly higher absolute weight gain during days 57-64 and 71-79, significantly lower absolute body weight gain during days 64-71 at high dose was observed. These significant differences were toxicologically not significant as total absolute gains during day 1-90 were comparable to vehicle control. at 450 mg/kg/day, increase in the weight of liver was associated with vacuolation of centrilobular hepatocytes in both sexes.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In males, the food consumption was significantly lower during Days 22-29, 29-36, 36-43, 50-57, 57-64, 71-79 and 79-85 was observed at mid dose. In females, significantly higher food consumption during Days 1-8 at low dose, during Days 1-8 and 85-90 at mid dose, during Days 79-85 at high dose and significantly lower during Days 43-50 at high dose was observed.
These significant differences were not considered toxicologically relevant as the body weights were not altered by the treatment.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmological examination was carried out with an ophthalmoscope prior to start of treatment and at the end of the treatment period did not reveal any abnormalities in the eyes of the experimental rats.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At 450 mg/kg/day, increase in the total leukocyte, neutrophil, lymphocyte and monocyte counts
were observed in females and correlated with inflammation observed in lungs histologically.
There were no test item-related changes in coagulation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 450 mg/kg/day in females, increase in total cholesterol, HDL cholesterol and triglyceride were correlated to the centrilobular vacuolation in liver microscopically. Decrease in total protein, albumin in both sexes; A/G ratio in females were observed without microscopic correlates.
At 75 and 150 mg/kg/day, increase in total cholesterol, HDL cholesterol in females were considered as test item-related changes without microscopic correlates.

Endocrine findings:

no effects observed

Description (incidence and severity):

The administration of the test item for 90 consecutive days through oral gavage to Wistar rats at 75, 150 and 450 mg/kg/day did not show any test item-related changes in thyroid profile.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The administration of the test item for 90 consecutive days through oral gavage to Wistar rats at 75, 150 and 450 mg/kg/day did not show any test item-related changes in urinalysis parameters.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Home cage and Handling observations: No treatment-related abnormalities were observed in any of the tested dose groups in either sex.
- Open field observations: No treatment-related abnormalities were observed in any of the doses tested in either sex.
- Sensory observations: No treatment-related abnormalities were observed in any of the doses tested in either sex.
- Motor Activity: There were no significant differences observed at all the tested doses in both sexes. Incidence of significantly lower distance travelled during interval 2 was observed at all treated groups in females. This significant difference was considered toxicologically not relevant as the total distance travelled was comparable the vehicle control.
- Neuromuscular observation:
- Landing hind limb footsplay: Significantly lower hindlimb footsplay was observed in high dose males. This significant difference was considered to toxicologically not significant as there were no changes observed in the home cage or open field observations. Further, there were no clinical signs observed during daily clinical observation.
- Grip strength: Significantly lower hindlimb grip strength at high dose in males and forelimb grip strength at mid dose in females was observed. These significant differences were considered toxicologically not significant as the animals were normal in the home cage and open field observations.
- Physiological observation:
- Body temperature: Incidence of significantly lower body temperature was observed in males at mid dose. This change was considered to be toxicologically not significant as there was no dose dependency.
- Body weight: There were no significant differences observed in the body weights, measured at the end of neurological observations at any of the doses tested in both sexes.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 450 mg/kg/day, increase in the weight of liver was associated with vacuolation of centrilobular
hepatocytes in both sexes.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross pathological findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings were observed in liver (centrilobular vacuolation of hepatocytes), lungs (increased alveolar macrophages/chronic inflammation), spleen (vacuolated macrophage in red pulp) and mandibular lymph nodes (vacuolated macrophage) in males and/or females.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Oestrous Cycle Evaluation: The vaginal smear was examined for all the animals prior to necropsy and following is the details of various stages observed. Most of the rats showed diestrous and proestrous to necropsy

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

450 mg/kg bw/day (nominal)

System:

immune system

Organ:

liver

lungs

lymph node

spleen

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Dose Formulation Analysis of the Test Item
The dose formulations were considered acceptable, as the percent agreement of the analyzed concentrations were in the range, 90% to 110% of the claimed concentrations and the relative standard deviation (RSD) was equal to or less than 10.0%.

 

Data tables

Table 1: Details of Experimental Design, Treatment Regime, Clinical Pathology Investigations, Sacrifice and Pathology Schedule.

 

				Clinical pathology investigations				Pathology
Group No.	Dose (mg/kg/ day)	No. of rats per Group	Treatment Period (Days 1-90)	Haema -tology	Coagula -tion	Clinical Chemistry	Urina -lysis	Gross Pathology	Organ Weights	Histopathology	Sacrificed on Day
G1	0	M:10 F: 10	M: + F: +	+	+	+	+	+	+	+	91
G2	75	M:10 F: 10	M: + F: +	+	+	+	+	+	+	X	91
G3	150	M:10 F: 10	M: + F: +	+	+	+	+	+	+	X	91
G4	450	M:10 F: 10	M: + F: +	+	+	+	+	+	+	+	91

+: Performed
M: Male
F: Female
X: Gross lesions

 

 

 

 

 

 

Table 2: Total leukocyte, neutrophil, lymphocyte and monocyte counts in males and females (percentage alterations).

 

Sex	Male			Female
Group	G2	G3	G4	G2	G3	G4
Dose (mg/kg/day)	75	150	450	75	150	450
No. of rats	10	10	10	10	10	10
WBC	―	―	―	―	―	↑(24)
Neut A	―	―	―	―	―	↑(55)*
Lymp A	―	―	―	―	―	↑(13)
Mono A	―	―	―	―	―	↑(46)

 

 

 

 

 

 

 

 

↑: Increased
*: Statistically significant
─: Not statistically/ biologically significant

Values in parenthesis indicate percentage change, when compared to vehicle control

 

 

 

 

 

 

 

 

Table 3:  Total cholesterol, HDL cholesterol, triglyceride, protein and albumin counts, and A/G ratio in males and females (percentage alterations).

 

Sex	Male			Female
Group	G2	G3	G4	G2	G3	G4
Dose (mg/kg/day)	75	150	450	75	150	450
No. of rats	10	10	10	10	10	10
T.Chol	―	―	―	↑(21)*	↑(20)*	↑(28)*
HDL Chol	―	―	―	↑(19)*	↑(15)*	↑(25)*
Trig	―	―	―	―	―	↑(129)*
T.Pro	―	―	↓(6)*	―	―	↓(9)*
ALB	―	―	↓(7)*	―	―	↓(23)*
A/G ratio	―	―	―	―	―	↓(27)*

 

 

 

 

 

 

 

 

 

 

↑: Increased ↓: Decrease *: Statistically significant

─: Not statistically/ biologically significant

Values in parenthesis indicate percentage change, when compared to vehicle control

 

 

Table 4: Increase in the weight of liver was associated with vacuolation of centrilobular hepatocytes in males and females (percentage alterations).

 

Sex	Male			Female
Group	G2	G3	G4	G2	G3	G4
Dose (mg/kg/day)	75	150	450	75	150	450
No. of rats	10	10	10	10	10	10
Liver     –Absolute	―	―	↑(6)	―	―	↑(27)*
–Relative to Bwt	―	―	↑(10)*	―	―	↑(28)*
–Relative to Brain wt	―	―	↑(8)	―	―	↑(29)*

↑: Increased *: Statistically significant ─: Not statistically/ biologically significant

Values in parenthesis indicate percentage change, when compared to vehicle control

 

 

Table 5: Treatment related microscopic findings observed in liver, lungs, spleen and mandibular lymph nodes in males and females.

 

Sex	Males				Females
Group	G1	G2	G3	G4	G1	G2	G3	G4
Dose (mg/kg/day)	0	75	150	450	0	75	150	450
No. of rats	10	10	10	10	10	10	10	10
LIVER; Vacuolation; hepatocyte; centrilobular minimal mild moderate	(10) 0 − − −	(10) 0 − − −	(10) 0 − − −	(10) 9 6 3 −	(10) 0 − − −	(10) 0 − − −	(10) 0 − − −	(10) 9 4 3 2
LUNGS (WITH BRONCHI AND BRONCHIOLES); Alveolar macrophages, increased minimal mild Inflammation; chronic; bronchioloalveolar; multi-focal minimal mild	(10) 0 − − 0 − −	(10) 4 4 − 0 − −	(10) 3 3 − 0 − −	(10) 2 2 − 0 − −	(10) 2 2 − 0 − −	(10) 2 2 − 0 − −	(10) 2 2 − 0 − −	(10) 10 6 4 5 2 3
SPLEEN; Vacuolation; macrophage; red pulp minimal mild	(10) 0 − −	(10) 0 − −	(10) 0 − −	(10) 9 8 1	(10) 0 − −	(10) 0 − −	(10) 0 − −	(10) 10 1 9
LYMPH NODES, MANDIBULAR; Vacuolation; macrophage; unilateral/bilateral minimal	(10) 0 −	(10) 0 −	(10) 0 −	(10) 1 1	(10) 0 −	(10) 0 −	(10) 0 −	(10) 9 9

 

Conclusions:

Considering the changes observed in haematology, clinical chemistry, organ weights and microscopic changes at 450 mg/kg/day, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of the test item is considered to be 150 mg/kg/day under the test conditions and doses employed.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-06-19 to 2012-11-13

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

no analytical verification of the dose was performed

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): JEFFCAT DPA
- Physical state: Liquid
- Lot/batch No.: # 1D516
- pH: 11.7
- Storage of the test item: The samples were stored in a specific room (Test Item Storage Room), at room temperature, protected from humidity and light; A sub-sample of the test item (Archival of Test Item) was preserved in the same condition as the main sample; The remaining amount of test item was disposed according to the Sponsor's instruction.

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BIOAGRI Laboratorias Ltda-DF
- Age at study initiation: 10 - 11 weeks old
- Housing: Each animal was housed individually, except during cohabitation. After acclimation, one male was placed into each female cage for pairing. After pairing, females that presented vaginal smears with the presence of sperm were considered mated and housed individually. The rats were housed in polypropylene cages (41 x 34 x 19 cm) with wire mesh tops and bedding material (wood shavings). Clean cages were provided twice weekly for all animals. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Upon receipt from the supplier, animals were placed into cages (1 animal/cage/sex) examined and acclimated for 7 days. All animals were observed for morbidity and mortality. At the end of this period, the animals were weighed and a detailed clinical examination was performed. Animals not selected were euthanized by inhalation of carbon dioxide and then exsanguinated. Only animals with body weights within ± 20% from the group mean body weight and with no abnormal signs were used in this study. In addition, all animals used in the study had a regular estrous cycle.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 - 23.6 °C
- Humidity (%): 40.7-70.0%
- Air changes (per hr): ambient room air will be changed 10-20 times per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Test solution was prepared daily and was used within 2 hours after preparation

For each dosage group, the appropriate amount of the test item was weighed into a pre-calibrated beaker. The vehicle (deionized water) was added in sufficient quantity to achieve the desired concentration. Each solution was stirred and dispensed into individual containers properly identified. A sufficient quantity of the vehicle was be similarly dispensed for administration to control animals. The prepared solutions were stored at room temperature.

VEHICLE:
- Amount of vehicle (if gavage): The volume administered each day was 4 mL/kg body weight.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Parental animals (males and females) were treated starting at 10-11 weeks old and ending when the animals were euthanized. Satellite animals (5 animals per sex of the control and 5 animals per sex of the high dose group) were kept for 14 days after the scheduled necropsy of parental animals without treatment, for observation of reversibility, persistence or delayed occurrence of toxic effects. Satellite animals were not mated and, consequently, were not used for assessment of reproduction/developmental toxicity.

Frequency of treatment:

7 days per week basis

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

nominal ingested

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

nominal ingested

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

nominal ingested

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

nominal ingested

No. of animals per sex per dose:

12 animals/sex/dose group (including vehicle group) + 5 animals/sex/satellite vehicle group + 5 animals/sex/500 mg/kg/day satellite group

Control animals:

yes, concurrent vehicle

Details on study design:

The following doses were chosen for this study:
- 10 mg/kg body weight/day as the expected dose which causes no signs;
- 100 mg/kg body weight/day as the intermediate dose level;
- 500 mg/kg body weight/day as the expected dose which causes signs of systemic toxicity, but not death or severe suffering.

Satellite animals (5 animals per sex of the control and 5 animals per sex of the high dose group) were kept for 14 days after the scheduled necropsy of parental animals without treatment, for observation of reversibility, persistence or delayed occurrence of toxic effects. Satellite animals were not mated and, consequently, were not used for assessment of reproduction/developmental toxicity.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
During premating, mating, gestation and lactation period, the animals were observed twice a day for morbidity, mortality and clinical observation for overt signs of ill health on working days and once a day on weekend and public holidays. These include, but are not limited to, changes in skin and fur, eye and mucous membranes, respiratory, circulatory, autonomic and central nervous system, motor activity and behavioral patterns. Animals found dead were necropsied. Weak or moribund animals were euthanized and necropsied. The carcasses were disposed in biological garbage and then incinerated.

BODY WEIGHT:
Males were weighed on the first day of dosing and weekly thereafter (including mating and post-mating periods). Females were weighed on the first day of dosing and once a week during premating and mating periods, on days 0, 7, 14 and 20 of gestation, and during lactation on the same days as weighing of litters (on days 0 and 4 postnatal).

FOOD CONSUMPTION:
Food consumption was determined on the same day of body weight determination (except on day 0) during premating and lactation periods. During gestation period food consumption was determined on days 3, 6, 9, 12, 15, 18 and 20. After the mating period, food consumption of males was determined weekly. Food consumption was not determined during mating period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
Hematology and clinical chemistry determinations were performed on 5 parental animals/sex/group, randomly selected from each group. The animals were fasted overnight and anesthetized by CO2 prior to blood collection (cardiac puncture). The following parameters were measured:
Red Blood Cell Count, hemoglobin, hematocrit, platelets, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, total white blood cell count, differential leukocyte count, band neutrophils, monocytes, segmented neutrophils, lymphocytes, eosinophils, basophils; clotting parameters: prothrombin time, activity partial thromboplastin time

CLINICAL CHEMISTRY: Yes
Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total protein, albumin, glucose, total cholesterol, urea nitrogen, creatinine, sodium, potassium, calcium, globulin, albumin/globulin ratio

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Sensory reactivity to stimuli and motor activity assessment were performed in 5 animals/sex/group. In males, these tests will be done at the end of the dosing period, before scheduled necropsy, and in females during lactation. The following parameters were assessed:
A - Autonomic functions: lacrimation, salivation, palpebral closure, prominence of the eye, piloerection, respiration;
B - Reactivity and sensitivity: sensor motor responses to approach tactile and tail flick;
C - Excitability: reactions to handling and behavior in an open field;
D - Gait and sensor motor coordination: degree of mobility and gait pattern in an open field;
E - Abnormal clinical signs: including convulsions, tremors, unusual behavior and deposits around the eyes, nose or mouth

Sacrifice and pathology:

- Gross necropsy
At termination all parental animals were examined macroscopically for any abnormalities or pathological changes. The animals were disposed in biological garbage and then incinerated.
For all animals a core list of organs was collected, weighed and preserved in 10% neutral formalin during necropsy.
Paired organs were weighed together.
- Histopathology:
At scheduled necropsy, the following organs of all animals were preserved: testes, epididymides, ovaries, prostate, seminal vesicle and coagulating gland, bulbourethral gland, organs showing alterations.
The following organs and tissues of 5 animals/sex/group were preserved: adrenals (right and left), bone marrow (femur), brain (cerebrum, cerebellum and pons), esophagus, heart, intestine (duodenum, jejunum, ileum - including Peyer's patches, cecum, colon, rectum/anus), kidneys (right and left), liver (3 lobes), lungs, lymph nodes (mesenteric and submaxillary), peripheral nerve (sciatic), spinal cord (cervical, midthoracic and lumbar sections); spleen, stomach (glandular and non-glandular), trachea, pancreas, thymus, thyroid/parathyroid, urinary bladder, uterus and all gross lesions.
Full histopathology of the preserved organs and tissues listed were performed in the highest dose and control animals. These examinations not extended to animals of other dosage groups, because treatment-related changes were not observed in the high dose group.

Other examinations:

Organ weights: At scheduled necropsy, testes and epididymides of all males were weighed.
Organ weights were obtained for the following organs from 5 animals/sex/group: liver, kidneys, adrenals, thymus, spleen, brain, heart

Statistics:

Quantitative variables such as body weights, food consumption and organ weights have been analyzed by One Way Analysis of Variance (ANOVA), followed by Dunnett's test if significance was detected, or by non-parametric test of Kruskal-Wallis, according to the results of tests for normality and homogeneity of variance. For qualitative or non-parametric data such as clinical findings, macroscopic and microscopic findings and fetal findings, comparison between means have been carried out using the Fischer's Exact Test or the Chi-square test. The level of significance was set at 5%.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Female No 105 presented respiratory sounds at day 6 of the test item administration; while female No 104 did not show any clinical sign and female No 116 had moderate dyspnea, piloerection and exophthalmos on day 7. Male No 109, at the same dose level, was euthanized for humane reason on day 8 of treatment, after severe signs of dyspnea, respiratory sounds, apathy and rhinodacryorrhea. Necropsy and histopathological examination of animals No 104, 105 and 109 showed pulmonary congestion and edema as result of bronchoaspiration by gavage error.

Male rats No 83, 86, 87, 92 and 94, exposed to 500 mg/kg/day presented moderate dyspnea and respiratory sounds between days 9 and 31 of test item administration; while males No 91 and 93 were noted with respiratory sounds on day 30 and 30-31, respectively. At the same dose level, male No 86 had piloerection and rhinodacryorrhea from day 13 to day 17. In females No 97, 101, and 102 was noted respiratory sounds from day 17 to day 27 of test item administration and also on day 43 of the test item administration (animal 97). Male No 92 and females No 102 and 106 had rhinodacryorrhea on days 10, 35 and 44 of treatment respectively. Finally, from day 17 to day 25 female No 97 showed moderate dyspnea; while female No 106 presented piloerection and slight apathy on days 43 and 44 of treatment.

Although these clinical signs were moderate in magnitude, they were sporadically observed throughout the dosing period in the test animals and were transitory in nature. Due to the extremely corrosive nature of the test substance to animal tissues and the dosing volumes used in the dosing of the high dose animals, it is likely that these respiratory observations were artefacts from the gavage dosing procedure. While these observations could be considered test item-related, they are not considered to be toxicologically relevant and are of low concern in the absence of other treatment related effects.

Mortality:

mortality observed, treatment-related

Description (incidence):

The test substance did not cause treatment-related mortality during the study. However, 3 females (No 104, 105 and 116) at 500 mg/kg/day were found dead on days 13, 7 and 7 of treatment, respectively.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No biologically or statistically significant changes were observed in body weight of treated males compared to the control group. Nevertheless, statistically higher body weight gain was observed in male rats exposed to 100 mg/kg/day from day 0 to day 7 (+ 1.8%); while at 500 mg/kg/day the difference was + 302.6%, from day 14 to day 21. Despite changes, these did not affect statistically overall body weight gain (from day 0 to day 28), although it was slightly lower in treated males when compared to the control group, but without a clear dose response relationship (-7.5% low, -8.3% mid and -8.2% high dose), and thus, were not considered related to treatment with test item.
During treatment and recovery period, mean body weight of treated satellite males was biologically and statistically unaffected by the treatment. However, overall body weight gain was statistically lower (-27%) during the treatment period (from day 0 to day 35); while in the recovery period, although without statistical significance overall body weight gain (from day 0 to day 14) was higher than control (+ 165.5%). In spite of these differences, these findings were not considered biologically relevant because in both cases body weight was unaffected by the treatment with the test item.
Statistically lower mean body weights were observed in female rats at 500 mg/kg/day on days 7 (-15.1%), 14 (-14.1%) and 20 (-19.3%) of the gestation period and on days 0 (-19.4%) and 4 (-17.5%) of the lactation period. Despite differences, these were not considered to be dose related, because at the study initiation the mean body weight of this group was lower than control (-7.7%) and then the differences during the gestation and lactation period were slight (<12%) and without a dose response relationship. During all treatment periods, the mean body weight gain of treated females was lower when compared to the control group, however, a dose related trend was not observed and these differences only were statistically significant at 500 mg/kg/day, affecting overall body weight gain (-34.1%) during the gestation period (from day 0 to day 20). Because body weight gain of treated females was erratic and without a dose related trend, these changes were not considered to be test item related.
At different observation intervals, body weight and body weight gain of treated satellite females showed statistically significant differences; however, these changes began at the study initiation when body weight of treated satellite females was lower than control (-17.6%) and accordingly it affected overall body weight and body weight gain during treatment and recovery period. Accordingly, changes observed in body weight and body weight gain were not considered to be test item related.
At different observation intervals, body weight and body weight gain of treated satellite females showed statistically significant differences; however, these changes began at the study initiation when body weight of treated satellite females was lower than control (-17.6%) and accordingly it did affect overall body weight and body weight gain during treatment and recovery period.
Accordingly, changes observed in body weight and body weight gain were not considered to be test item related.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No biologically or statistically significant changes were observed in food consumption of treated males when compared to the control group.
Statistically significant lower food consumption was observed in females exposed to 500 mg/kg/day on days 0 to 7 (-13.5%) and on days 0 to 14 (-11.3%) of the premating period. During the gestation period, statistically lower food consumption was observed on days 3 to 6 (-14.7%) and on days 18 to 20 (-5%) at 500 mg/kg/day; while at 100 mg/kg/day statistically lower food consumption was noted on days 9 to 12 (-6.4%) and on days 15 to 18 (-0.6%) compared to the control group. These differences were small in magnitude and not dose related and thus, were not considered to be test item related.
At treatment initiation (from day 0 to day 7) food consumption of treated satellite females was statistically lower (-28.4%) compared to the control group, affecting statistically overall food consumption (from day 0 to day 35) -12.1%. Because the difference was very slight, it was not considered to be treatment-related.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant higher red blood cell count (+1.7%) was observed in male rats at 10 mg/kg/day; while male rats exposed to 500 mg/kg/day showed statistically lower mean corpuscular hemoglobin concentration (-3.2%). Because the differences were very small in magnitude and without a dose related trend, these findings were not considered to be treatment related.
Females at 500 mg/kg/day displayed statistically lower mean corpuscular values (-5.3%) and mean corpuscular hemoglobin (-7%) and females exposed to 10 mg/kg/day had statistically lower mean corpuscular hemoglobin (-4%). These changes were isolated and very small in magnitude and therefore, not considered toxicologically relevant. In treated satellite females, statistically significant lower platelets (-22%) was observed, but this value was only slightly lower than Bioagri's Historical Data Base (553.5-696.9) and is without toxicological significance.
In male rats exposed to 10 mg/kg/day was noted statistically lower lymphocytes (-0.8%), while at 500 mg/kg/day was observed statistically higher monocytes (+120%) and eosinophils (+40%). Despite these differences, in all cases they occurred without a dose response relationship and are not considered to be related to treatment with the test item.
Statistically significant lower segmented neutrophils were observed in females exposed to 500 mg/kg/day (-57.5%) with a dose related trend; however, the values are within of Bioagri's Historical Data Base (325.4-1217) and therefore was considered as not toxicologically relevant in absence of the control group, but without a dose response relationship and then not considered to be test item related. Statistically lower segmented neutrophils (-46.6%), monocytes (-16.7%) and eosinophils (-2.6%) were observed in treated satellite females, but these values are within Bioagri's Historical Data Base and are without toxicological significance.
No biologically or statistically significant changes were observed in clotting parameters of treated males and females compared to the control group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant higher sodium (+10%) at 500 mg/kg/day and aspartate aminotransferase at 100 and 500 mg/kg/day (+6.4% and 3.6%, respectively) were observed in male rats when compared to the control group. These differences were considered as not biologically relevant, because in both cases were small in magnitude and a dose response relationship has not been observed;
Treated satellite males showed statistically higher alanine aminotransferase (+35.2%) but within laboratories' historical database and not considered to be treatment related.
In females exposed to 500 mg/kg/day statistically significant higher glucose (+44.5%) and albumin/globulin ratio (+14.3%) was noted compared to the control group. However, in the first case no dose related and in the second one in small magnitude and therefore not considered to be test item related. A very small difference in mean sodium was noted in females at 10 mg/kg/day (+0.4%) compared to the control group, although it was considered to be an incidental finding.
Statistically significant higher urea nitrogen (+59.1%), total protein (+68.6%), globulin (+63.2%), albumin (+75%), calcium (+57.4%), cholesterol (+112.3%) and potassium (+63.8%) were observed in treated satellite females. The changes, while statistically significant, were not observed in any other high dose group (male or female) and were not supported by other pathology findings. Also, the values were within the laboratories' historical database and these findings were not considered to be biologically relevant, and not test item related.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observational battery:
Male No 83 at 500 mg/kg/day presented moderate dyspnea and respiratory sounds; while female No 97 at the same dose level had respiratory sounds. Despite that these findings were observed at the highest dose level, they occurred only in two animals, without statistical significance. No other findings related to the sensory reactivity to stimuli and motor activity assessment was noted.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Adrenal weight was statistically lower in males exposed to 500 mg/kg/day (-24.3%) compared to the control group. Relative to body weight, although without statistical significance, adrenal weight was lower than control (-13.6%), being statistically lower relative to brain weight (22.7%). Despite differences, these were considered as not biologically significant, because they were moderate in magnitude and also no macroscopic or microscopic lesions were observed. Also, heart weight relative to body weight was statistically higher at 100 mg/kg/day compared to the control group (+18.6%), although it was considered incidental.
Treated satellite males showed statistically higher kidney weights relative to brain weight (+31.8%) when compared to the control group. However, in absence of statistical significance in kidney weight and kidney weights relative to body weight and because no macroscopic or microscopic lesions were observed, this finding was not considered to be treatment related.
Statistically lower heart weight was noted in female rats at 10 and 500 mg/kg/day (-18.6% and -20.4%, respectively) compared to the control group. However, no statistical differences were observed in heart weight relative to body weight, and relative to brain weight only were observed statistical significance at 10 mg/kg/day. In addition, at 500 mg/kg/day spleen and brain weights were statistically lower than control (-34.9% and -7.3%, respectively). Nevertheless, relative to body weight, liver and brain weights were statistically higher (+12.5% and +19.8%, respectively). Finally, relative to brain weight spleen and brain weights were statistically lower (-29.5% and -7.3%, respectively) when compared to the control group. Because in all cases the differences were moderate in magnitude and without a dose response relationship, this finding was not considered related to treatment with the test item.
Statistically significant lower heart (-20.4%), kidney (-33%) and brain (-6.3%) weights were observed in treated satellite females when compared to the control group. Nevertheless, relative to body weight, these organs did not show statistical significant and relative to brain weight only was observed in kidney weight (-28.4%). Also, in these animals, adrenal weights, relative to body weight (+29%) and relative to brain weight (+22.8%) was statistically significant compared to the control group. Despite changes observed, these were not considered to be treatment related, because were moderate in magnitude and no macroscopic or microscopic lesions were observed.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related findings were observed upon necropsy in male or female rats. Animals No 104, 105 and 109 exposed to 500 mg/kg/day, found dead or euthanized for human reasons during the study showed macroscopic lesions or bronchoaspiration, compatible with gavage error and confirmed during histopathological examination.
Although pulmonary emphysema (bilateral), in female No 116 at 500 mg/kg/day was observed, this lesion was not found in other treated animals. Despite clinical signs observed in both male and female rats exposed to 500 mg/kg/day during treatment period, no macroscopic lesions were found, except in male No 83, where pulmonary congestion was observed.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related microscopic changes were noted in male or female rats.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

> 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Male systemic toxicity, maternal systemic toxicity

Critical effects observed:

no

In this study, the only findings considered to be treatment-related were certain clinical signs observed only in male and female rats at high dose level. While these findings were likely attributable to the dosing of the test article, they were not considered to be of toxicological significance.

Although these clinical signs were moderate in magnitude, they were sporadically observed throughout the dosing period in the test animals and were transitory in nature. Due to the corrosive nature of the test substance to animal tissues and the dosing volumes used in the dosing of the high dose animals, it is likely that these respiratory observations were artefacts from the gavage dosing procedure. While these observations could be considered test item-related, they are not considered to be toxicologically relevant and were of low concern in the absence of other findings related to male sytemic toxicity, maternal systemic toxicity, and embryo-fetal toxicity.

Conclusions:

Under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test item in Wistar rats was greater than 500 mg/kg/day for male systemic toxicity and maternal systemic toxicity.

Executive summary:

The test substance was tested in rats according to OECD 422 guideline. The substance was dosed at 0, 10, 100, 500 mg/kg/day. No treatment-related mortality during the study was reported. Most male and female rats exposed to 500 mg/kg/day presented respiratory clinical signs between treatment days 6 to 44. Although these clinical signs were moderate in magnitude, they were sporadically observed throughout the dosing period in the test animals and were transitory in nature. Due to the extremely corrosive nature of the test substance to animal tissues and the dosing volumes used in the dosing of the high dose animals, it is likely that these respiratory observations were artefacts from the gavage dosing procedure. While these observations could be considered test item-related, they are not considered to be toxicologically relevant and are of low concern in the absence of other findings related to male systemic toxicity, maternal systemic toxicity, and embryo-fetal toxicity.

No treatment-related changes in body weight and body weight gain, food consumption, haematology, clinical chemistry, neurobehavioural changes, organ weight, macroscopic and microscopical (histophatological) evaluation of organs were reported.

Under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test item in Wistar rats was greater than 500 mg/kg/day for male systemic toxicity and maternal systemic toxicity. For risk assessment purposes, 500 mg/kg bw/day is selected as NOAEL value.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP-compliant, guideline study

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

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:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

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:

short-term repeated dose toxicity: dermal

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 - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

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

Additional information

Repeated toxicity: oral

Short-term study: A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed in rats, in which male and female rats were exposed to 0 (vehicle), 10, 100, 500 mg/kg bw/d via gavage (OECD 422). The vehicle used was water and the test solutions were prepared daily and administered within 2 hours after preparation. The test substance did not cause mortality (treatment-related) during the study; however, male and female rats exposed to 500 mg/kg/day presented respiratory clinical signs between treatment days 6 to 44. Also, during the functional observational battery, in one male and one female rat at the same dose level, dyspnea and respiratory sounds were observed.

Although these clinical signs were moderate in magnitude, they were sporadically observed throughout the dosing period in the test animals and were transitory in nature. Due to the corrosive nature of the test substance to animal tissues and the dosing volumes used in the dosing of the high dose animals, it is likely that these respiratory observations were artefacts from the gavage dosing procedure. While these observations could be considered test item-related, they are not considered to be toxicologically relevant and are of low concern in the absence of other findings related to male systemic toxicity and maternal systemic toxicity. Based on the abovementioned considerations, the NOAEL was considered to be > 500 mg/kg bw/day (nominal dose received) for male and systemic toxicity and maternal systemic toxicity, and greater than 500 mg/kg bw/day for embryo-fetal toxicity.

 

Longer-term study: A 90-day repeated dose toxicity study (K1, OECD 408; Malleshappa, 2021) was performed in male and female Wistar rats. The test item was dissolved in Milli-Q® Water and administered to rats at the graduated dose levels of 0 (vehicle), 75, 150 and 450 mg/kg/day. The rats in the vehicle control group (G1) group received vehicle Milli-Q® Water alone. The dose volume administered was 10 mL/kg body weight. Each group in the experiment was comprised of ten male and ten female rats. No mortality was observed at any dose tested. Transient clinical sign of slight salivation was observed in all animals at 450 mg/kg/day soon after the dose administration.  The incidence of post-dosing salivation observed in 450 mg/kg/day may be attributed to oral mucosa irritation, caused by the administration of test item. In the present study there were no gross or microscopic changes in gastrointestinal tract. Therefore, it was considered that the transient salivation observed in this study was of no toxicological significance.

At 450 mg/kg/day test item-related adverse effects were observed in both sexes. Haematological parameters revealed, increase in the total leukocyte, neutrophil, lymphocyte and monocyte counts in females. These findings were considered as the secondary changes associated with test item related inflammation in lungs. Clinical chemistry parameters showed, increase in total cholesterol, HDL cholesterol and triglyceride in females were correlated to the centrilobular vacuolation in liver microscopically. Decrease in total protein, albumin in both sexes; A/G ratio in females were observed without microscopic correlates. Organ weights showed, increase in the weight of liver was associated with vacuolation of centrilobular hepatocytes in both sexes. Increased incidences/severity of alveolar macrophages/chronic inflammation in lungs, vacuolated macrophages in red pulp of spleen and vacuolated macrophages in mandibular lymph nodes were noted in either males and/or females. The chronic inflammation of lungs was characterized by infiltration of mixed inflammatory cells in perivascular and peribronchial area with thickening of alveolar septa along with increased alveolar macrophages and considered as adverse. The alveolar macrophages were present in the form of multifocal small collections of macrophages with medium to abundant foamy cytoplasm, particularly in peribronchial and/or sub-pleural areas.

Increase in total cholesterol, HDL cholesterol in females were also observed at 75 and 150 mg/kg/day. These changes were considered as test item-related non-adverse as no microscopic correlates. Microscopically, minimal to moderate degree vacuolation was noted in liver at 450 mg/kg/day in both sexes. However, the hepatocyte vacuolation did not induce inflammation or necrotic changes or did not show any changes in liver enzyme profile and thus considered as non adverse. The increased liver weight and severity/ incidences of hepatocyte vacuolation were more in females as compared to males. No gross findings were observed. Considering the changes observed in haematology, clinical chemistry, organ weights and microscopic changes at 450 mg/kg/day, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of the test item is considered to be 150 mg/kg/day.

 

Repeated toxicity: inhalation

A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the inhalation route of exposure.

 

Repeated toxicity: dermal

A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the dermal route of exposure.

 

Justification for classification or non-classification

Based on the available data and according to the criteria of the CLP Regulation, the test substance should not be classified for STOT repeated exposure via the oral route.