Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Description of key information

There were no toxicologically significant changes in the examined parameters (clinical signs, body weight and body weight gain, food consumption, hematology, blood coagulation and clinical chemistry, necropsy findings, organ weights) after the 14-day oral (by gavage) administration of 100, 300 mg/kg bw/day doses.

Main study OECD 422 in progress (05/2019), preliminary result available (NOAEL: 600 mg/ kg bw/d). Based on available data (no adverse effects observed in pre study OECD 407) the substance is not classified with repeated dose toxicity according to the CLP regulation 1272/2008/EC.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 July - 09 November, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
03 October 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3050 Repeated Dose 28–Day Oral Toxicity Study in Rodents, July 2000.
Version / remarks:
July, 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
yes
Species:
rat
Strain:
Wistar
Remarks:
Hsd.Han: of Wistar origin
Details on species / strain selection:
Species / Strain: Rat, Hsd.Han: of Wistar origin

The rat is commonly used species for toxicological studies in accordance with international recommendations. The Wistar rat was the system of choice because it has been the preferred and most commonly used species for oral toxicity tests is a well-known laboratory model with sufficient historical data.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species / Strain: Rat, Hsd.Han: of Wistar origin
Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90.
Hygienic level: SPF (Specific pathogen-free) at arrival and kept in good conventional environment during the study.
Age of animals at start of Male animals: 53 – 58 days
the study: Female animals: 53 – 58 days
Body weights at start of 233 – 259 g for male animals
the study: 147 – 170 g for female animals
The weight variation did not exceed ¿ 20 per cent of the mean weight
Number and sex of animals: 20 naïve male and 20 naïve female (nulliparous and non-pregnant animals) rats
Number of groups: 4 (3 dose levels + 1 control group)
Number of animals/group: 10 (5 male; 5 female)
Animal health: Only healthy animals were used for the study. Healthy status was certified by the breeder.
Acclimatization time: 8 days

Husbandry

Housing conditions

Animal room no.: 18/2
Housing: 5 animals of the same sex/ cage
Cage type: Type IV polypropylene/ polycarbonate

Bedding: Certified laboratory wood bedding (Lignocel¿Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG; D-73494 Rosenberg Holzmühle 1 Germany; see Appendix 12). The cages and bedding were changed twice a week.
Illumination: Artificial light, from 6 a.m. to 6 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: Above 10 air-exchanges/ hour by a central air-condition system.
Environmental conditions were maintained by an air-conditioning system. Temperature and relative humidity were verified and recorded daily during the study.

Animals received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany and tap water, as for human consumption, ad libitum except overnight food deprivation before the blood sampling.
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used.
Animals received tap water from watering bottles. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Váci út 172-174. Budapest, H-1138 Hungary). The quality control results are available at Toxi-Coop Zrt.’s archives.

Identification of animals

Individual identification was performed by numbers on the tail of the animals written with a permanent marker. In the pre-treatment period, the numbers were given on the basis of the laboratory master file of Toxi-Coop Zrt. and were be re-marked as necessary to ensure correct identification.

Identification numbers of animals
GROUPS DOSE
(mg/kg bw/day) MALES FEMALES
Group 1 0 6549 6576
6551 6586
6562 6592
6567 6593
6572 6595
Group 2 50 6550 6582
6556 6583
6557 6585
6564 6591
6569 6597

GROUPS DOSE
(mg/kg bw/day) MALES FEMALES
Group 3 100 6553 6577
6554 6578
6563 6587
6570 6589
6573 6598
Group 4 300 6552 6575
6558 6580
6566 6584
6568 6588
6571 6596

The cages were marked by identity cards, with information about the study number, duration of the study, name of the test item, dose and mode of administration, species and strain, sex of animals, cage number and individual animal numbers, start of the administration, date of the necropsy. Boxes were arranged in such a way that possible effects due to cage placement are minimized.

Randomization

Animals were randomly assigned to test groups. All animals were sorted according to body weight by computer and grouped according to weight ranges. There were an equal number of animals from each weight group in each of the experimental groups during the randomization. The grouping was controlled by SPSS/PC computer program according to the actual body weight verifying the homogeneity and deviations among the groups and cages.
Route of administration:
oral: gavage
Details on route of administration:
The test item was administered orally via gavage. The route of application was selected in compliance with international guidelines . The oral route is the possible route of human exposure to the test item.
Vehicle:
water
Details on oral exposure:
The test item was orally administered daily (7 days per week) in graduated doses to three groups of experimental animals for a period of 14 days. Control animals were treated concurrently with the vehicle only. The actual treatment volume was calculated according to the most recent body weight. A treatment
volume of 5 mL/kg body weight was applied. Animals were not treated on the day of gross pathology.

A control and three dose groups were involved in the study.
The dose setting with 0, 100, 300 and 1000 mg/kg bw/day is based on the literary data of p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) and in agreement with the Sponsor. Doses are selected with the aim of inducing toxic effects but no mortality or suffering at the highest dose and a NOAEL at the lowest dose.

Groups Dose(mg/kg bw day) Dose volume(mL/kg bw) Number of animals
Male Female
Group 1 0 5 5 5
Group 2 100 5 5 5
Group 3 300 5 5 5
Group 4 1000 5 5 5
Remark: Group 1 = Control; Group 2 = Low dose; Group 3 = Mid dose;
Group 4 = High dose;
Animals in Group 1 only received the vehicle.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration check of the formulated test item

Analytical control of dosing formulations was performed once during this DRF study. Five aliquots of 5 mL of each formulation to be administered to the animals (20, 60 and 200 mg/mL) and five aliquots of 5 mL control substance (vehicle) were taken.

Sampling date: July 25, 2017
Date of analysis: July 25, 2017

The p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) concentrations in the samples varied within the range from 103 % to 109 % in comparison to the nominal values.
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. The recovery of the test item from the vehicle was within the acceptance criteria (relative to nominal concentrations: 104 % at ca. 1 mg/mL and 104 % at ca. 200 mg/mL).
p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) proved to be stable in distilled water at the intended concentrations at room temperature for three days. A separate analytical report (Study no. 805-100-2254) provided these data.
Duration of treatment / exposure:
The experimental period involved 8 days of acclimatization, 14 days treatment and observation period and necropsy on the following day (study Day 14).
The day of first treatment was considered as Day 0 of examination.

Frequency of treatment:
once a day
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Number of animals/group: 10 (5 male; 5 female)
Control animals:
yes, concurrent vehicle
Details on study design:
The dose setting with 0, 100, 300 and 1000 mg/kg bw/day was based on the literary data of p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) and in agreement with the Sponsor.
Four groups of Hsd.Han: of Wistar rats consisting of five animals per group and sex were administered orally (by gavage) once daily at 0 (vehicle only), 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/day) doses in nominal concentrations of 0, 20, 60 and 200 mg/mL corresponding to a 5 mL/kg bw dosing volume. A group of vehicle (distilled water) treated animals (n= 5/sex) served as a control.
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front.
The p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) concentrations in the samples used for administration of animals varied within the range from 103 % to 109 % in comparison to the nominal values, thereby confirming proper dosing.
Detailed clinical observations were performed once daily after the treatment. Body weights were recorded once weekly. The food consumption was determined weekly to coincide with body weight measurements during the study. Clinical pathology (hematology, blood coagulation and clinical chemistry) and gross pathology examinations were conducted on all animals one day after the last treatment (on Day 14). Selected organs were weighed.

Observations and examinations performed and frequency:
Clinical observations

Detailed clinical observations were conducted once a day, after treatment at approximately the same time. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling.
Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

Body weight and body weight gain

Individual body weights were recorded on Day 0 (prior to study start) and once weekly (on Days 7, and 13) with a precision of 1 g. Individual body weight changes were calculated according to the days of measurements and for the study overall.
The animals were also weighed immediately prior to sacrifice (on Day 14; fasted body weight) in order to calculate organ weight to body weight ratio.

Food consumption measurement

Food consumption was determined with the measurement of non-consumed diet with a precision of 1 g once weekly to coincide with body weight measurements (given food on Days 0 and 7; remained food on Days 7 and 13). All animals were food deprived overnight prior to blood sampling.

Hematology, blood coagulation and clinical biochemistry

Clinical pathology examinations including hematology, blood coagulation and clinical chemistry were conducted at termination of the treatment (i.e. one day after the last treatment).
Animals were food deprived overnight (for approximately 16 hours) prior to blood collection. Blood samples were harvested from the retro orbital venous plexus under Isofluran CP® anesthesia. Three samples were taken from each animal: one for hematology (MiniCollect®K3EDTA tubes, spray-dried, 0.25 mL), one for determination of blood clotting times (MiniCollect® 3.8 % sodium citrate, for APTT and PT 1.0 mL) and the third one (Vacuette 2.5 mL Z Serum Sep C/A) to obtain serum samples for clinical chemistry. All of the three tubes are manufactured by Greiner Bio-One International AG, Kremsmünster, Austria.
The tubes for hematology and coagulation were filled up to the final volume (marked on the tubes) and at least 1.0 mL blood was collected into the clinical chemistry tubes.

Sacrifice and pathology:
Pathology
Necropsy

Gross pathology was performed on every experimental animal at termination of the treatment i.e. one day after the last treatment, on Day 14.
Animals were anesthetized with Isofluran CP® and were exsanguinated from the abdominal aorta after verification of deep narcosis.
The external appearance (surface of the body, all orifices) was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically for each animal. All observations were recorded with details of the location, color, shape and size.
The following organs/tissues were removed and preserved in 4 % formaldehyde solution, except testes, epididymides, which were preserved in modified Davidson solution and then stored in 4 % formaldehyde solution for possible future histopathological examination:


List of organs preserved
Adrenal glands
Aorta
Bone marrow (femur)
Brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata)
Eyes (lachrymal gland with Harderian glands and optic nerve)
Female mammary gland
Heart
Kidneys
Large intestines (caecum, colon, rectum, including Peyer’s patches),
Liver
Lungs (with main stem bronchi; inflation with fixative and then immersion ;)
Lymph nodes (submandibular, mesenteric)
Muscle (quadriceps)
Esophagus
Pancreas
Pituitary
Prostate
Salivary glands (submandibular)
Sciatic nerve
Seminal vesicle with coagulating gland
Sexual organs: testes with epididymides , seminal vesicles with coagulating gland, ovaries, uterus with uterine cervix and oviducts, vagina
Skin
Small intestines (representative regions: duodenum, ileum, jejunum)
Spinal cord (at three levels: cervical, mid-thoracic and lumbar)
Spleen
Sternum
Stomach
Thymus
Thyroid + parathyroid
Trachea
Urinary bladder
* Thyroid and parathyroid were preserved together with pharynx
Organs and tissues were excised, trimmed of any adherent tissue, as appropriate, weighed, and preserved as described above.

Organ weight

The following organs were weighed and recorded. Paired organs were weighed together.
With precision of 0.01g: Liver, kidneys, testes, epididymides, seminal vesicles with coagulating gland and prostate as a whole, uterus with fallopian tubes, thymus, spleen, brain and heart.
With precision of 0.001g: Adrenal glands


Histopathology

Histopathological examinations were not performed as dose selection for the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat (main study) was possible with the results of this 14-day dose range finder study.

Statistics:
Statistical analysis was done with SPSS PC+ software for the following data:
- Body weight
- Hematology
- Blood coagulation
- Clinical chemistry
- Organ weight

The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.
Frequency of clinical signs and pathological findings by sex and dose was calculated.
The use of the word “significant” or “significantly” indicates a statistically significant difference between the control and the experimental groups. Significance was judged at a probability value of p < 0.05 and < 0.01. Male and female rats were evaluated separately.
Clinical signs:
no effects observed
Description (incidence and severity):
P-Nitrobenzoic Acid, Compound with 2,2’,2”-Nitrilotriethanol (1:1) caused no clinical signs at 100, 300 or 1000 mg/kg bw/day in male or female animals during the two weeks treatment period.
Mortality:
no mortality observed
Description (incidence):
There was no mortality during the course of the study (control 100, 300 and 1000 mg/kg bw/day).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight gain was slightly depressed in male and female animals at 1000 mg/kg bw/day on week 1 resulting in a slightly lower body weight in male and female animals on Day 7 and in male animals on Day 13.

At 100, 300 mg/kg bw/day, there were no toxicologically significant changes in body weight and body weight gain.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean daily food consumption was slightly reduced in male and female animals at 1000 mg/kg bw/day between Days 0 and 7 in full compliance with the body weight changes.
The mean daily food consumption was similar in the control and 100 mg/kg bw/day and 300 mg/kg bw/day test item treated animal (male and female) during the 14-day observation period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Hematological evaluation revealed test item related changes in some of the examined red blood cell parameters (lower red blood cell count, hemoglobin concentration, hematocrit mean corpuscular hemoglobin concentration, or elevated mean corpuscular volume) and elevated percentage of reticulocytes in male and female animals at 1000 mg/kg bw/day coinciding with necropsy observation and changes in organ weights.
At 100, 300 mg/kg bw/day, there were no toxicologically significant changes in hematology, blood coagulation after the 14-day oral (by gavage) administration.

Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Pathological test item effects were not detected upon the evaluation of the clinical chemistry parameters in male or female animals at 100, 300 or 1000 mg/kg bw/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The behavior and physical condition of animals were normal during the14-day treatment period.
Immunological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day:
mean albumin: globulin ratio (A/G) were slightly but statistically significantly higher than in the control group in male animals of high dose group.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Slightly but statistically significantly higher mean spleen weights (absolute and relative to body and brain weights) were indicative of a test item influence both in male and female animals at 1000 mg/kg bw/day and were coinciding with changes in hematology parameters and necropsy observations.
100 mg/kg bw/day
Statistically significant difference with respect to the control was detected at the lower mean thymus weight (absolute) in male animals.
The weights of examined organs (absolute and relative to body and brain weights) were comparable with their control in the female animals of low dose group.
300 mg/kg bw/day
The mean thymus weights (absolute and relative to body and brain weights) were lower than in the control group in male animals.
In the female animals, there were no statistically significant differences with respect to their control in the examined organ weights (absolute and relative to body and brain weights).
1000 mg/kg bw/day
Statistically significant difference with respect to the control was observed at the lower mean fasted body weight, at higher mean spleen weights (absolute and relative to body and brain weights) and at the lower mean weights of adrenal glands (absolute and relative to brain weight) in male animals. The mean weights of liver, kidneys and testes each relative to body weight exceeded the control value.
In the female animals, the mean spleen weights (absolute and relative to body and brain weights) exceeded the control value but reaching statistical significance at spleen weight relative to body weight. Some statistically significant difference with respect to the control was noted for the higher mean brain weight relative to body weight and for the lower mean fasted bodyweight relative to brain weight due to the slight body weight changes. The mean weights of adrenal glands (absolute and relative to brain weight) were lower than in the control group.
The changes in weight of these organs (liver, kidneys, thymus, adrenal glands and testes) were with minor degree and there were no related macroscopic findings. These were considered to be of little or no biological significance and histological examinations could reveal the nature of these changes.

The statistically significant differences with respect to the control in liver, kidneys and testes weights were mainly due to the lower mean fasted body weight of male animals at 1000 mg/kg bw/day as statistical differences ceased when referring to the brain weight. Dose related changes in the thymus weight might be indicative of normal but accelerated involution. Histological examinations are needed to reveal the nature of this findings.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Necropsy observation revealed enlargement and dark color of the spleen, which were induced by the treatment with test item in male and female animals at 1000 mg/kg bw/day in accordance with the observation at hematological and organ weight evaluations.

Control group
One side pyelectasia in the kidney was observed in one control male animal (1/5).
In the female control group, dark colored liver (1/5), point-like hemorrhages in the thymus (1/5) and slight hydrometra (1/5) were seen at the necropsy.
100 mg/kg bw/day
In on male animal administered with the low dose, yellowish gray foci in one side epididymidis (1/5) were seen.
Slight or moderate hydrometra was observed in female animals (2/5).
300 mg/kg bw/day
In one male animal, point-like hemorrhages were observed in the thymus (1/5).
Marked hydrometra was noted for single female animal (1/5).
1000 mg/kg bw/day
Necropsy observations revealed enlarged spleen (3/5), dark colored spleen (1/5), yellowish gray foci in one side epididymidis (1/5) and point-like hemorrhages in the thymus (1/5) in male animals of high dose group at the necropsy.
In the female animals, enlarged spleen (1/5), dark colored spleen (3/5) and slight hydrometra (1/5) were observed at the necropsy.

Dilatation of renal pelvis (pyelectasia) and yellowish gray foci in the epididymidis are common individual changes in untreated experimental rats. Both of these findings were seen irrespective to doses in this study thus test item influence might be excluded.
Thymic hemorrhage is frequently observed in experimental rats and was considered to be related to the exsanguination procedure i.e. it was consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination. Similarly, dark colored liver was consequence of exsanguination procedure.
Hydrometra, related to the female sexual cycle, is also a frequent observation in experimental rats. In the lack of related inflammatory or other pathological signs hydrometra was judged to be toxicologically not relevant.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
Histopathological examinations were not performed as dose selection for the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat (main study) was possible with the results of this 14-day dose range finder study.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
Mortality

There was no mortality in the control, 100, 300 or 1000 mg/kg bw/day groups during the
14-day treatment period (male and female).

Clinical observations

p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) caused no clinical signs in female animals at 100, 300 or 1000 mg/kg bw/day in male or female. The behavior and physical condition of animals were normal during the14-day treatment period.

Body weight and body weight gain

The body weight development was slightly and transiently depressed in male and female animals at 1000 mg/kg bw/day on week 1.
100 mg/kg bw/day and 300 mg/kg bw/day
There were no significant differences between the control and test item treated male animals in the mean body weight during the 14-day observation period. Although the mean body weight gain was slightly below the relevant control value during the two weeks – no statistical significances – the mean body weight was comparable in the control and test item treated groups on each measuring day.
1000 mg/kg bw/day
The body weight gain was slightly lower than in the control group in male and female animals of high dose group between Days 0 and 7 and in male animals for the study overall (between Days 0 and 13). This slight but statistically significant reduction in body weight gain resulted in lower mean body weight on Day 7 (male and female) and on Day 13 (male).


Food consumption

The mean daily food consumption was slightly reduced in male and female animals at 1000 mg/kg bw/day between Days 0 and 7 in full compliance with the body weight changes.
The mean daily food consumption was similar in the control and 100 mg/kg bw/day and 300 mg/kg bw/day test item treated animal (male and female) during the 14-day observation period.


Hematology

Hematological evaluation revealed slight test item related changes in some of the examined red blood cell parameters (lower mean red blood cell count, hemoglobin concentration. hematocrit value or mean corpuscular hemoglobin concentration, higher mean corpuscular volume) and elevated percentage of the reticulocytes at 1000 mg/kg bw/day (male and female).

100 mg/kg bw/day
In the male animals of low dose group, statistical significance was observed at the higher mean percentage of neutrophil granulocytes (NEU) along with a lower mean percentage of lymphocytes (LYM) with respect to the control.
The examined hematological parameters were comparable in the female control and test item treated groups.
300 mg/kg bw/day
Slight but statistically significant differences with respect to their controls were noted in male animals at the lower mean percentage of eosinophil granulocytes (EOS), at lower mean corpuscular hemoglobin concentration (MCHC) and at the slightly longer mean prothrombin time (PT).
In the female animals, the mean percentage of basophil granulocytes (BASO) was slightly but statistically significantly lower than in the control.
1000 mg/kg bw/day
Statistically significant differences with respect to their control were noted for the higher mean percentage of neutrophil granulocytes and reticulocytes (RET) and for the lower mean percentage of eosinophil granulocytes, red blood cell count (RBC), hemoglobin concentration (HGB) and mean corpuscular hemoglobin concentration. The mean corpuscular volume (MCV) was higher and the mean prothrombin time was longer than in the control group in male animals of the high dose group comparing to the control.
In the female animals of high dose group, lower mean percentage of eosinophil granulocytes and basophil granulocytes, lower mean red blood cell count, lower mean hemoglobin concentration, hematocrit (HCT) value and lower mean corpuscular hemoglobin concentration were observed when comparing to their control. The mean mean corpuscular volume and mean percentage of reticulocytes exceeded the control value in female animals of the high dose group.
A test item influence was supposed in development of the lower mean red blood cell parameters (red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume or mean corpuscular hemoglobin concentration) and elevated percentage of reticulocytes both in male and female animals administered with 1000 mg/kg bw/day.
The other slight but statistically significant differences with respect to control were considered to have no or minor toxicological relevance as these were at a low magnitude or were not related to doses (NEU, LYM, EOS, BASO and PT) and all values remained well within the historical control ranges.

Clinical chemistry

Pathological test item effects were not detected upon the evaluation of the clinical chemistry parameters in male or female animals at 100, 300 or 1000 mg/kg bw/day.
100 mg/kg bw/day
In the male animals, there was no statistically significant difference with respect to their control in the examined clinical chemistry parameters.
In the female animals of low dose group, statistical significance was observed at the slightly lower mean concentration of calcium (Ca2+) and potassium (K+) with respect to the control.

300 mg/kg bw/day
The mean concentration of total bilirubin (TBIL) was slightly higher than in the control in male animals.
In the female animals, there were no statistically significant differences between the control and 300 mg/kg bw/day treated groups in the examined clinical chemistry parameters.
1000 mg/kg bw/day
The mean activity of alanine amino transferase (ALT), creatinine (CREA) concentration and mean albumin: globulin ratio (A/G) were slightly but statistically significantly higher than in the control group in male animals of high dose group.
In the female animals, lower mean concentration of potassium with respect to the control was detected when compared to the control.
The slight elevation of alanine amino transferase activity (male animals at 1000 mg/kg) might be indicative of the test item on hepatic function.
Although the differences between the control and test item treated groups were statistically significant, there was no dose relevance for most of the above mentioned parameters (TBIL, K+, Ca2+) and values of these parameters remained well within the historical control ranges. Therefore changes in clinical chemistry parameters were judged to be toxicologically negligible.

Necropsy

Necropsy observation revealed enlargement and dark color of the spleen, which were induced by the treatment with test item in male and female animals at 1000 mg/kg bw/day in accordance with the observation at hematological and organ weight evaluations.

Control group
One side pyelectasia in the kidney was observed in one control male animal (1/5).
In the female control group, dark colored liver (1/5), point-like hemorrhages in the thymus (1/5) and slight hydrometra (1/5) were seen at the necropsy.
100 mg/kg bw/day
In on male animal administered with the low dose, yellowish gray foci in one side epididymidis (1/5) were seen.
Slight or moderate hydrometra was observed in female animals (2/5).
300 mg/kg bw/day
In one male animal, point-like hemorrhages were observed in the thymus (1/5).
Marked hydrometra was noted for single female animal (1/5).
1000 mg/kg bw/day
Necropsy observations revealed enlarged spleen (3/5), dark colored spleen (1/5), yellowish gray foci in one side epididymidis (1/5) and point-like hemorrhages in the thymus (1/5) in male animals of high dose group at the necropsy.
In the female animals, enlarged spleen (1/5), dark colored spleen (3/5) and slight hydrometra (1/5) were observed at the necropsy.

Dilatation of renal pelvis (pyelectasia) and yellowish gray foci in the epididymidis are common individual changes in untreated experimental rats. Both of these findings were seen irrespective to doses in this study thus test item influence might be excluded.
Thymic hemorrhage is frequently observed in experimental rats and was considered to be related to the exsanguination procedure i.e. it was consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination. Similarly, dark colored liver was consequence of exsanguination procedure.
Hydrometra, related to the female sexual cycle, is also a frequent observation in experimental rats. In the lack of related inflammatory or other pathological signs hydrometra was judged to be toxicologically not relevant.


Organ weight

Slightly but statistically significantly higher mean spleen weights (absolute and relative to body and brain weights) were indicative of a test item influence both in male and female animals at 1000 mg/kg bw/day and were coinciding with changes in hematology parameters and necropsy observations.
100 mg/kg bw/day
Statistically significant difference with respect to the control was detected at the lower mean thymus weight (absolute) in male animals.
The weights of examined organs (absolute and relative to body and brain weights) were comparable with their control in the female animals of low dose group.
300 mg/kg bw/day
The mean thymus weights (absolute and relative to body and brain weights) were lower than in the control group in male animals.
In the female animals, there were no statistically significant differences with respect to their control in the examined organ weights (absolute and relative to body and brain weights).
1000 mg/kg bw/day
Statistically significant difference with respect to the control was observed at the lower mean fasted body weight, at higher mean spleen weights (absolute and relative to body and brain weights) and at the lower mean weights of adrenal glands (absolute and relative to brain weight) in male animals. The mean weights of liver, kidneys and testes each relative to body weight exceeded the control value.
In the female animals, the mean spleen weights (absolute and relative to body and brain weights) exceeded the control value but reaching statistical significance at spleen weight relative to body weight. Some statistically significant difference with respect to the control was noted for the higher mean brain weight relative to body weight and for the lower mean fasted bodyweight relative to brain weight due to the slight body weight changes. The mean weights of adrenal glands (absolute and relative to brain weight) were lower than in the control group.
The changes in weight of these organs (liver, kidneys, thymus, adrenal glands and testes) were with minor degree and there were no related macroscopic findings. These were considered to be of little or no biological significance and histological examinations could reveal the nature of these changes.

The statistically significant differences with respect to the control in liver, kidneys and testes weights were mainly due to the lower mean fasted body weight of male animals at 1000 mg/kg bw/day as statistical differences ceased when referring to the brain weight. Dose related changes in the thymus weight might be indicative of normal but accelerated involution. Histological examinations are needed to reveal the nature of this findings.
Key result
Dose descriptor:
dose level:
Effect level:
>= 100 - <= 300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
immunology
organ weights and organ / body weight ratios
Remarks on result:
other: no toxicologically significant changes in the examined parameters (clinical signs, body weight- body weight gain, food cons., hemat., blood coag., clin. chem, necropsy, organ weights) after the 14-day oral administration of 100, 300 mg/kg bw/day doses.
Key result
Dose descriptor:
dose level:
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other: slight and transient depression of body weight and food consumption, changes in hematological parameters, macroscopic and weight changes in spleen in male- female Wistar rats after the consecutive 14-day oral administration at 1000 mg/kg bw/day.
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
immune system
Organ:
spleen
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
immune system
Organ:
thymus
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
endocrine system
Organ:
adrenal glands
testes
Conclusions:
p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) caused slight and transient depression of body weight and food consumption, changes in hematological parameters (red blood cell parameters and reticulocytes) macroscopic and weight changes in spleen (enlarged and dark colored, elevated weight of spleen) in male or female Hsd.Han: Wistar rats after the consecutive 14-day oral (by gavage) administration at 1000 mg/kg bw/day.

There were no toxicologically significant changes in the examined parameters (clinical signs, body weight and body weight gain, food consumption, hematology, blood coagulation and clinical chemistry, necropsy findings, organ weights) after the 14-day oral (by gavage) administration of 100, 300 mg/kg bw/day doses.

Based on the observations made in this toxicity study, the dose levels for a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat (main study) were determined as follows:

Group 1 Vehicle control
Group 2 60 mg/kg bw/day
Group 3 200 mg/kg bw/day
Group 4 600 mg/kg bw/day
Executive summary:

The objective of this study was to obtain first information on the toxic potential ofp-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)in rats at three dose levels to allow a dose-setting for a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat (main study).The dose setting with 0, 100, 300 and 1000 mg/kg bw/day was based on theliterary data ofp-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)and in agreement with the Sponsor.

Four groups of Hsd.Han: of Wistar rats consisting of five animals per group and sex were administered orally (by gavage) once daily at 0 (vehicle only), 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/day) doses in nominal concentrations of 0, 20, 60 and 200 mg/mL corresponding to a 5 mL/kg bw dosing volume. A groupof vehicle (distilled water) treated animals (n= 5/sex) served as a control.

The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front.

The p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) concentrations in the samples used for administration of animals varied within the range from 103 %to109 %in comparison to the nominal values, thereby confirming proper dosing.

Detailed clinical observationswere performed once daily after the treatment.Body weights were recorded once weekly. The food consumption was determined weekly to coincide with body weight measurements during the study. Clinical pathology (hematology, blood coagulation and clinical chemistry) and gross pathology examinations were conducted on all animals one day after the last treatment (on Day 14). Selected organs were weighed.

The results of this study were summarized as follows:

Mortality:There was no mortality during the course of the study (control 100, 300 and 1000 mg/kg bw/day).

 

Clinical observations:P-Nitrobenzoic Acid, Compound with 2,2’,2”-Nitrilotriethanol (1:1) caused no clinical signs at 100, 300 or 1000 mg/kg bw/dayin male or female animals during the two weeks treatment period.

 

Body weight and body weight gain: The body weight gain was slightly depressed in male and female animals at 1000 mg/kg bw/day on week 1 resulting in a slightly lower body weight in male and female animals on Day 7 and in male animals on Day 13.

 

Food consumption:The mean daily food consumption was slightly reduced in male and female animals at 1000 mg/kg bw/day between Days 0 and 7 in full compliance with the body weight changes


Hematology and blood coagulation:Hematological evaluation revealed test item related changes in some of the examined red blood cell parameters (lower red blood cell count, hemoglobin concentration, hematocrit mean corpuscular hemoglobin concentration, or elevated mean corpuscular volume) and elevated percentage of reticulocytes in male and female animals at 1000 mg/kg bw/daycoinciding with necropsy observation and changes in organ weights.

 

Clinical chemistry:Pathological test item effects were not detected upon the evaluation of the clinical chemistry parameters in male or female animals at 100, 300 or 1000 mg/kg bw/day.

 

Necropsy: Test itemrelated enlargement and dark color of the spleenwere observedin male and female animals at 1000 mg/kg bw/dayat the necropsy.

 

Organ weight: The higher mean spleen weights(absolute and relative to body and brain weights)were indicative of a test item influence both in male and female animalsat 1000 mg/kg bw/day and were in accordance with changes in hematology parameters and necropsy observations.

 

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018 - study in progress
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
yes
Route of administration:
oral: gavage
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Key result
Dose descriptor:
NOAEL
Effect level:
> 600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Remarks on result:
other: preliminary results, study in progress
Key result
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

Justification for classification or non-classification