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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
In addition to the required examinations, special attention was given to the reproductive organs of male and female animals.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Name of test substance: Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen
CAS No. 13897-55-7 and 13897-56-8
Test substance No: 10/0117-5
Batch identification: B4360
Purity: 99.762%
Homogeneity: given (study code 15L00360)
Stability: stable until 21 Jul 2017

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
SELECTION OF DOSES / CONCENTRATIONS
In a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422; 85R0117/10S114; supporting study) the oral administration of the test substance by gavage to male and female Wistar rats resulted in severe clinical findings at a dose level of 250 mg/kg bw/d (reduced food consumption and body weight). Although the dose level was reduced to 200 mg/kg bw/d from study day 7 onwards, clinical finings remained. In addition, a single female animal treated at 100 mg/kg bw/d died prematurely. The cause of death was not possible to assess.

Therefore, the following dose levels were selected:
100 mg/kg bw/d as high dose
25 mg/kg bw/d as intermediate dose
5 mg/kg bw/d as low dose

The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
Mortality
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

Clinical observations
All animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal.

Detailed clinical observations
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined:
1. Abnormal behavior when handled
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Impairment of gait
12. Lacrimation
13. Palpebral closure
14. Exophthalmus
15. Feces (appearance/ consistency)
16. Urine
17. Pupil size

Food consumption
Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.

Water consumption
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

Body weight data
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

Functional observational battery
A functional observational battery (FOB) was performed in all animals at the end of the administration period starting at about 10:00 h. At least one hour before the start of the FOB the rats were transferred to single-animal polycarbonate cages. Drinking water was provided ad libitum, but no food was offered during the measurements. The FOB started with passive observations without disturbing the rats, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

Home cage observations:
The animals were observed in their closed home cages; during this period any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the rats. Attention was paid to:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Gait abnormalities
6. Other findings

Open field observations:
The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined:
1. Behavior on removal from the cage
2. Fur
3. Skin
4. Salivation
5. Nasal discharge
6. Lacrimation
7. Eyes/ pupil size
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/ stereotypes
14. Gait abnormalities
15. Activity/ arousal level
16. Feces excreted within 2 minutes (appearance/ consistency)
17. Urine excreted within 2 minutes (amount/ color)
18. Rearing within 2 minutes
19. Other findings

Sensory motor tests/ reflexes:
The animals were then removed from the open field and subjected to following sensory motor or reflex tests:
1. Reaction to an object being moved towards the face (approach response)
2. Touch sensitivity (touch response)
3. Vision (visual placing response)
4. Pupillary reflex
5. Pinna reflex
6. Audition (auditory startle response)
7. Coordination of movements (righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (tail pinch)
11. Grip strength of forelimbs
12. Grip strength of hindlimbs
13. Landing foot-splay test
14. Other findings

Motor activity assessment
Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the rats were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts was counted over 12 intervals for 5 minutes per interval. The sequence in which the rats were placed in the cages was selected at random. On account of the time needed to place the rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the rats during these measurements and the measurement room was darkened after the transfer of the last rat. The program requires a file name for the measured data to be stored. This name consists of the reference number and a serial number.

Ophthalmoscopy
Prior to the start of the administration period on day -3 the eyes of all animals and on study day 84 the eyes of the control and high-dose animals were examined for any changes using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt, Germany).

Estrous cycle determination
Estrous cycle length and normality were evaluated daily for all female animals for a minimum of 3 weeks prior to necropsy.
Sacrifice and pathology:
CLINICAL PATHOLOGY
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence.
The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
The results of clinical pathology examinations were expressed in Inter¬national System (SI) units.
The following examinations were carried out in all animals per test group and sex at the end of the administration period.

PATHOLOGY
Necropsy
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

Organ weights
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (terminal body weight)
2. Adrenal glands
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Ovaries
10. Pituitary gland
11. Prostate
12. Seminal vesicle with coagulating glands
13. Spleen
14. Testes
15. Thymus
16. Thyroid glands
17. Uterus with cervix

Organ/ tissue fixation
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymis, left (modified Davidson’s solution)
12. Esophagus
13. Extraorbital lacrimal glands
14. Eyes with optic nerve (modified Davidson’s solution)
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum (with Peyer’s patches)
20. Kidneys
21. Larynx
22. Liver
23. Lungs
24. Lymph nodes (mesenteric and axillary lymph nodes)
25. Mammary gland (male and female)
26. Nose (nasal cavity)
27. Ovaries
28. Oviducts
29. Pancreas
30. Parathyroid glands
31. Pharynx
32. Pituitary gland
33. Prostate
34. Rectum
35. Salivary glands (mandibular and sublingual glands)
36. Sciatic nerve
37. Seminal vesicles
38. Skeletal muscle
39. Skin
40. Spinal cord (cervical, thoracic and lumbar cord)
41. Spleen
42. Sternum with marrow
43. Stomach (forestomach and glandular stomach)
44. Testis, left (modified Davidson’s solution)
45. Thymus
46. Thyroid glands
47. Trachea
48. Urinary bladder
49. Uterus
50. Vagina

The left testis and left epididymis of all animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis was used for sperm parameters.
Statistics:
Body weight, body weight change: a comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means

Rearing, grip strength forelimbs, grip strength hindlimbs, foot-splay test, motor activity, estrous cycle: non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal medians

Organ weight parameters: non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No test substance-related, adverse effects were obtained in any test groups (5, 25 and 100 mg/kg bw/d).
Salivation after treatment was observed in 9 male and 1 female animals of test group 3 (100 mg/kg bw/d) on several days of the study, beginning on study day 55. From the temporary, short appearance immediately after dosing it was concluded that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Not treatment-related changes for body weight parameters occurred in male and female animals of test groups 1-3 (5, 25 and 100 mg/kg bw/d).

Mean body weight of females in test group 1 (5 mg/kg bw/d) on study day 63 was significantly increased.
Mean body weight change values were significantly increased in female animals of test group 1 (5 mg/kg bw/d) on study days 35 and 63.
With regard to these changes, a clear dose-response relationship did not occur over the complete course of treatment. Therefore, these changes were assessed as being incidental and not related to treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Female animal No. 52 showed scar on the upper edge of the right eyeball.

All apparent findings were assessed as being incidental in nature since they occurred in control as well as in treated animals and did not show a dose-response relationship.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
After three months of compound administration in males of test group 2 (25 mg/kg bw/d) relative reticulocyte counts were lower compared to controls, but the change was not dose-dependent. Therefore, this alteration was regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
After three months of compound administration, in males of test group 3 (100 mg/kg bw/d) potassium levels were decreased, but the values were within the historical control range (males, potassium: 4.26-5.04 mmol/L). Therefore, this change was regarded as incidental and not treatment-related.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Deviation from "zero values" was obtained in one female rat of test group 1 (5 mg/kg bw/d). However, this finding was without a dose-response relationship and occurred in single rat only, this observation was considered to have been incidental.
The following examinations were performed during FOB and have to be assessed individually:

Home cage observations:
No test substance-related effects were observed.

Open field observations:
No test substance-related effects were observed.

Sensorimotor tests/reflexes:
No test substance-related effects were observed.
Female animal No. 52 of test group 1 (5 mg/kg bw/d) showed scar in the right eye (upper edge). The finding was assessed to be spontaneous in nature and not related to treatment.

Quantitative parameters:
No test substance-related effects were observed.

Motor activity measurement
Regarding the overall motor activity as well as single intervals, no test substance-related deviations to the control animals were noted for male and female animals of test groups 1-3 (5, 25 and 100 mg/kg bw/d).
At interval No. 8, a decreased value was measured for male animals of test group 2 (25 mg/kg bw/d), and at interval No. 10, a decreased value was measured for male animals of test group 1 (5 mg/kg bw/d). The individual deviations were assessed not to be related to treatment as no dose-response relationship occurred.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
When compared with the control group 0 (set to 100%), the following mean absolute and relative weights were significantly increased or decreased in one or more test groups:

The significantly increased absolute (0.418 g) and relative (0.119%) mean weights of the cauda epididymis in test group 3 males were within the historical control range (0.410-0.454 g; 0.112-0.128%). The absolute and relative weights of the epididymis were not significantly changed and there were no treatment-related histopathological findings. Therefore, the increased weight of the cauda epididymis was regarded to be incidental.

The increased mean relative liver weight (2.532%) and the increased mean relative kidney weight (0.741%) in females of test group 3 were within the historical control range (liver: 2.162-2.723%, kidneys: 0.618-0.794%) and there were no histopathological correlates. Therefore, these weight increases were assessed to be incidental.

The mean absolute weights of the pituitary gland were significantly increased in females of test groups 1 and 2. The mean relative weights of the pituitary gland were significantly increased in test groups 1 and 3. Because there was no dose-response relationship and there was no histopathological correlate, the increased weights were considered to be incidental.

The mean absolute thyroid weights were significantly decreased in males of test group 1 (20.9 mg), test group 2 (19.9 mg), and test group 3 (20.1 mg). The mean relative thyroids weights were significantly decreased in test groups 1 (0.006%) and 2 (0.005%). The decreased thyroid weights were within the historical control range (17.0-28.8 mg / 0.005-0.008%), there was no dose-response relationship, and there were no treatment-related histopathological findings. Therefore, the decreased thyroid weights were regarded to be incidental.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings were single observations. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Estrous cycle
No test substance-related effects on estrous cycle length and the number of cycles were obtained.

Sperm parameters
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The oral administration of Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen by gavage to male and female Wistar rats for 3 months caused no adverse signs of toxicity in male and female animals at 100 mg/kg bw/d. Therefore, the no observed adverse effect level (NOAEL) was set to 100 mg/kg bw/d for male and female Wistar rats.
Executive summary:

Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 5 (test group 1), 25 (test group 2) and 100 mg/kg bw/d (test group 3) over a period of 3 months. With regard to clinical examinations, no signs of general systemic toxicity were observed even at a dose level of 100 mg/kg bw/d. In addition, no test substance-related effects on estrous cycle length and the number of cycles were obtained. Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose level of the compound of 100 mg/kg bw/d. Regarding pathology, there were no treatment-related organ weight changes, gross lesions, and histopathological findings in male and female Wistar rats. All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.