N,N''-(4-methyl-m-phenylene)bis[N',N'-dimethylurea]

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-08-16 to 2023-03-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(Adopted: 25 June 2018)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N''-(4-methyl-m-phenylene)bis[N',N'-dimethylurea]

EC Number:

241-523-6

EC Name:

N,N''-(4-methyl-m-phenylene)bis[N',N'-dimethylurea]

Cas Number:

17526-94-2

Molecular formula:

C13H20N4O2

IUPAC Name:

1-{5-[(dimethylcarbamoyl)amino]-2-methylphenyl}-3,3-dimethylurea

impurity 1

Reference substance name:

N,N''-(2-methyl-m-phenylene)bis[N',N'-dimethylurea]

Cas Number:

17607-23-7

Molecular formula:

C13H20N4O2

IUPAC Name:

N,N''-(2-methyl-m-phenylene)bis[N',N'-dimethylurea]

Specific details on test material used for the study:

- Name of the Test Item in study report: TDI-Urone 80
- Type of Formulation: Technical

SOURCE OF TEST MATERIAL
- Source: Alzchem Trostberg GmbH
- Batch number of test material: 022704
- Purity: 2,4-TDI Uron: 78.6 (% w/w), 2,6-TDI Uron: 20.8 (% w/w)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Dose formulation was achieved by mixing the test item. Solubility check was performed using the test item prior to initiation of dosing period at the desired concentration. Based on solubility distilled water was selected as vehicle. Test item formulations were prepared daily prior to the administration.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

(Crl:WI)

Details on species / strain selection:

The rat is preferred species by OECD TG 408 and has been historically proven to be a suitable test system for toxicity studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Bio-Technology (India) Pvt. Ltd. No. 4B MN Park, Turkapally Village, Shameerpet Mandal, Medchal District, Hyderabad, Telangana 500 078
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: males: 252.5 gram (Range: 224.0 to 281.1 gram) , females: 192.7 gram (Range: 171.2 to 215.2 gram)
- Fasting period before study: no
- Housing: Rats in groups of two/three were housed in clean, sterilized solid floored standard polypropylene rat cages covered with stainless steel grill. Cages were placed on stainless steel racks. Clean and sterilized corn cob was used as bedding material. Cage rotation was done at weekly intervals
- Diet (ad libitum): conventional laboratory rodent pellet feed (manufactured by Special Diet Services - England, supplied by Vivo Biotech Ltd., Hyderabad, India)
- Water (ad libitum): potable well water (IIBAT) processed through reverse osmosis
- Acclimation period: male rats were acclimatized for at a period of seven days and female rats were acclimatized for a period of nine days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0 – 21.9
- Humidity (%):45 - 59
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: Males from: 2021-08-24 To: 2021-11-21 and recovery To: 2021-12-20
Females from: 2021-08-26 To: 2021-11-24 and recovery To: 2021-12-22

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral route was chosen, as it is one of the possible routes of repeated exposure to human users, bystanders and probably also to domestic animals.

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

- Amount of vehicle (if gavage): 10 ml/kg b.w.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item formulation was subjected to analysis using HPLC in the analytical chemistry department, IIBAT for homogeneity, stability and active ingredient analysis once prior to the initiation of dosing. The sampling was performed from the top, middle and bottom of the dose formulations in duplicate preparations. The analysis was performed by validated analytical method at the Analytical Chemistry Department of IIBAT.
The mean recovery of the homogeneity, stability and active ingredient analysis of the test item was within the acceptable limits of 96.13%-106.22% recovery of the desired dose concentrations.
The active ingredient of TDI-Urone 80 in the various doses (250, 500 & 1000 mg/kg b.w.) was assayed prior to the initiation of dosing, the mean for top, middle and bottom samples of G2(250 mg/kg b.w.), G3(500 mg/kg b.w.) and G4 (1000 mg/kg b.w.) was 96.73%, 98.48% 106.11% respectively.

The mean recovery of stability at 0 hours of G2 low dose (250 mg/kg b.w.), G3 intermediate dose (500 mg/kg b.w.) and G4 high dose (1000 mg/kg b.w.) was 96.68, 100.04 and 103.49 respectively.

The mean recovery of stability at 4 hours of G2 low dose (250 mg/kg b.w.), G3 intermediate dose (500 mg/kg b.w.) and G4 high dose (1000 mg/kg b.w.) was 96.13, 98.17 and 104.70 respectively

Duration of treatment / exposure:

90 days

Frequency of treatment:

The test item was administered orally once daily for a period of 90 days using a stainless steel ball tipped oral intubation needle

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 (10 males and 10 females)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of the dose range finding study (Sponsor study no. 41104304)
- Fasting period before blood sampling for clinical biochemistry: yes
- Post-exposure recovery period in satellite groups: An additional 10 animals (five males and five females) were included in G1R and G4R dose groups for determining reversibility or persistence of toxicity in a minimum treatment-free period of 28 days from the day of last administration of test item.
- Dose range finding studies: yes

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily until the end of scheduled treatment and recovery period for all the animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the initiation of treatment and thereafter weekly for all animals till the end of scheduled sacrifice

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the administration of the test item (Day 1), weekly thereafter during the entire observation period and at terminal sacrifice on day 91 for main groups and on day 119 for recovery groups.

FOOD CONSUMPTION AND COMPOUND INTAKE
Recorded cage wise daily and reported on weekly basis.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the initiation of dosing and at the end of the treatment period for main groups and at the end of recovery period for recovery groups.
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from animals (G1 - G4) at the end of 90 day treatment (on Day 91) and in recovery groups (G1R and G4R) at the end of 28 days recovery period (on Day 119).
- Anaesthetic used for blood collection: Yes (isoflurane inhalation)
- Animals fasted: Yes (overnight fasted (water ad libitum))
- How many animals: all (10 male /10 female/groups)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected from animals (G1 - G4) at the end of 90 day treatment (on Day 91) and in recovery groups (G1R and G4R) at the end of 28 days recovery period (on Day 119).
- Animals fasted: Yes (overnight fasted (water ad libitum))
- How many animals: all (10 male /10 female/groups)

PLASMA/SERUM HORMONES: Yes (thyroid hormones triiodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH))
- Time of blood sample collection: at termination for main groups on day 91 and recovery groups on day 119
- How many animals: all surviving animals

URINALYSIS: Yes
- Time schedule for collection of urine: from all surviving animals at the end of the treatment period (Day 90) for main groups and at the end of the recovery period (Day 118) for recovery groups
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in the last week of treatment for the animals of the main groups (G1 to G4) and during the last week of recovery for the recovery groups (G1R and G4R).
- Dose groups that were examined: all
- Battery of functions tested: approach response, visual placing, touch response, click response, tail pinch response, toe pinch response, palpebral reflex, oculocardiac response, pupil light response, limb rotation, righting reflex, tail – limb reflex, positional passivity, positive geotropism, body temperature, abdominal muscle tone, limb muscle tone, landing foot splay, assessment of grip strength (forelimb and hind limb) and locomotor activity (30 min duration (3 cycles of 10 min each)).

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Gross necropsy was performed on all animals of main groups (G1 – G4) at the end of treatment period and on recovery group animals (G1R and G4R) at the completion of 28 days recovery period. Animals were humanely euthanized by carbon dioxide asphyxiation and subjected to detailed examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. Moribund or animals showing severe pain or enduring signs of severe distress was humanely euthanized when noticed and subjected to necropsy. Animals that are found dead during observation period was sent for necropsy immediately and examined grossly
for abnormalities, if any.

Organ weighing was performed under wet conditions as soon as possible after dissection for liver, kidneys, adrenals, testes, epididymides, prostate and seminal vesicles with coagulating glands as a whole, uterus, ovaries, thymus, spleen, brain, heart, pituitary gland and thyroid gland of all experimental groups.

HISTOPATHOLOGY: Yes (high dose and control group. Examinations were not extended to animals of other dose groups, as treatment-related changes are not observed in the high dose group)
- The following tissues were preserved in 10% neutral buffered formalin for histopathological examination: All gross lesions, brain (representative regions including cerebrum, cerebellum and medulla/pons), spinal cord (at three levels: cervical, mid-thoracic and lumbar), pituitary, thyroid with parathyroid, thymus, oesophagus, salivary glands, stomach, small and large intestines (including Peyer’s patches), liver, pancreas, kidneys, adrenals, spleen, heart, trachea and lungs (preserved by inflation with fixative and then immersion), aorta, testes, ovaries, uterus, cervix, vagina, ventral and dorsolateral prostate, epididymides, skin with mammary gland (male and female), urinary bladder, lymph nodes (mesenteric and mandibular), peripheral nerves (sciatic and tibial), muscle (Gastrocnemius), femur bone marrow.

Other examinations:

Vaginal smear was prepared from female animals on the day of terminal sacrifice just before euthanasia to determine the stages of estrous cycle

Statistics:

Numerical data obtained during the conduct of the study was subjected to calculation of group means and standard deviations and reported. Continuous data such as body weight, feed consumption, clinical pathology and organ weights of the main group animals (G1-G4) were analyzed for normality using Shapiro-Wilk test and homogeneity of variance using Levene's test. Homogeneous data were subjected to Analysis of Variance (ANOVA) followed by Dunnett's test for post hoc comparison. For non-homogenous data, a non-parametric test for comparing several groups, the Kruskal-Wallis test were performed. If significant difference is observed in the Kruskal-Wallis test, Mann-Whitney U test was performed to identify the group which showed a significant difference from control.
The data from the recovery groups (G1R and G4R) were subjected to normality and homogeneity and a parametric Student's t-test was performed to find out significant difference between the groups. In the case of non-normal distribution of data, the Mann-Whitney U test was used to compare the groups.
A p-value of < 0.05 was considered statistically significant. The analysis was performed using IBM SPSS Statistics Software version 28.
Non numerical data obtained during the conduct of the study was reported as individual results or incidences.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No test item related clinical signs of toxicity were observed in any of the animals in the treated group during the treatment and post treatment i.e. recovery period. All the animals were apparently normal from the initiation of dosing until scheduled sacrifice.

Mortality:

no mortality observed

Description (incidence):

No test item related morbidity/mortality were observed in any of the animals in the treated group during the treatment and post treatment i.e. recovery period. All the animals were apparently normal from the initiation of dosing until scheduled sacrifice.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no statistically significant test item related change in the body weight of the treated groups of both sexes when compared with the concurrent control group (for more details, please refer to Tables 1 and 2 in section "Any other information on results incl. tables")..

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no statistically significant test item related change in the feed consumption of the treated groups of both sexes when compared with the concurrent control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmological examination of the animals performed prior to the administration of the test item did not reveal any abnormalities. Similarly, no ocular lesions or changes were noticed in the treated animals during the ophthalmological examination performed at the end of the treatment and recovery period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase (40.20%) in clotting time (CT) in G2 male was observed when compared with respective control. On day 119, RBC in G4R male decreased (-7.36%) statistically significant when compared with respective control. Statistically significant increase in monocyte (44.54%) in G2 female was observed when compared with respective control. These changes were considered as incidental since their occurrence was sporadic with no dose dependency and not test item related. All the other parameters of treatment group animals were comparable with the respective control group.
No statistically significant test item related changes were observed in treated group animals in the coagulation parameters (APTT, PTT) in both the sexes when compared with respective control group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The G3 (-11.35%) and G4 (-18.83%) females exhibited statistically significant decrease in HDL, and significant increase (36.31%) was observed in the values of LDL in G4 female when compared with respective control. On day 119, creatinine (-5.71%) and chloride (-1.32%) in G4R female were decreased significantly when compared with respective controls. Significant decrease (-0.77%) was observed in Cl in G4R male. These changes were considered as incidental since their occurrence was sporadic with no dose dependency and not test item related. All the other parameters of treatment group animals were comparable with the respective control group.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

The plasma was analyzed for thyroid hormones triiodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH) using ELISA method. Statistically significant increase was observed in the values of T3, in G2 (62.96%) and G3 (33.33%) male when compared with respective control. These changes in T3 values coincided with increase in thyroid organ weights in G2 and G3 males. However, no statistically significant changes were observed in high dose group male as well as female animals. On day 119, TSH in G4R male decreased (-15.00%) statistically significant when compared with respective control. Hence these changes were considered as incidental since their occurrence was sporadic with no dose dependency (for more details, please refer to Tables 3 and 4 in section "Any other information on results incl. tables").

Urinalysis findings:

no effects observed

Description (incidence and severity):

The results of urinalysis performed at the end of treatment (G1 - G4) and recovery period (G1R and G4R) did not reveal any treatment related changes in either sex when compared with respective controls.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decrease in fore limp grip strength was observed in G2 (-14.69%) and G3 (-15.87%) males when compared with the control group.
A statistically significant decrease was observed in G2 (-13.22%), G3 (-17.91%) and G4 (-24.87%) females in case of fore limp grip strength when compared with the control group. These changes were considered as dose dependent. However, these changes disappeared in the recovery group. Moreover, none of the animals exhibited any clinical signs related to neuromuscular function. In addition, there was no statistical difference observed in high dose male animals and therefore these changes were considered as transient effects. All the other parameters of treatment group animals were comparable with the respective control group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was a statistically significant increase in the relative weights of the epididymides in G2 (15.20%), G4 (9.31%) males and thyroid in G2 (12.68%) males when compared with respective control. However, in females there was a statistically significant decrease (-12.39%) in the relative weights of brain in G4R when compared with control recovery. Although these observed changes were statistically significant, they did not occur in a dose dependent manner, hence considered as not related to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross pathological changes (abnormalities) were observed in control and treated group animals. No congenital /incidental findings were observed in the gross pathology in the control and treated group

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item related histopathological changes were observed in the treated group animals. No incidental findings were observed in the histopathology of the organs/tissues of the control and treated group animals.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

The vaginal smears were collected on the day of sacrifice from all females of G1, G2, G3, G4 of 10 females in each group G1R and G4R of 5 females per group.The vaginal smear estrous cycle evaluation was in correlation with the vaginal cytology of respective groups.

Details on results:

No gross pathological changes (abnormalities) were observed in control and treated group animals. No congenital /incidental findings were observed in the gross pathology in the control and treated group

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 1 Summary of body weight (g) – male

DAYS
GROUP/DOSE (mg/kg b.w.)	1	8	15	22	29	36	43	50	57	64	71	78	^@85	^@90
G1 Control	252.73	310.15	363.90	408.47	444.36	474.19	503.42	522.88	548.38	558.94	576.17	589.15	606.39	614.09
	±	±	±	±	±	±	±	±	±	±	±	±	±	±
	17.76	17.89	19.96	24.01	27.06	27.79	29.17	29.10	31.17	31.90	33.11	34.76	37.16	36.14
G2 Low dose 250	251.94	307.86	355.77	395.66	427.88	454.43	477.60	499.30	515.47	531.89	546.70	561.49	576.06	581.82
	±	±	±	±	±	±	±	±	±	±	±	±	±	±
	13.26	14.02	16.38	16.53	17.97	18.35	23.43	25.95	29.77	30.80	33.24	36.62	39.38	40.44
% Change from Control	-0.31	-0.74	-2.24	-3.14	-3.71	-4.17	-5.13	-4.51	-6.00	-4.84	-5.11	-4.70	-5.00	-5.25
G3 Intermediate dose 500	253.46	304.07	351.94	390.98	421.97	455.17	481.43	505.06	524.10	544.74	560.25	572.25	583.44	590.79
	±	±	±	±	±	±	±	±	±	±	±	±	±	±
	13.88	17.40	22.16	28.46	32.47	39.46	40.54	41.23	44.55	43.03	46.92	49.31	52.99	52.04
% Change from Control	0.29	-1.96	-3.29	-4.28	-5.04	-4.01	-4.37	-3.41	-4.43	-2.54	-2.76	-2.87	-3.78	-3.79
G4 High dose 1000	253.12	310.45	362.41	404.77	438.73	470.73	500.95	519.49	538.62	556.41	573.31	588.01	603.13	608.86
	±	±	±	±	±	±	±	±	±	±	±	±	±	±
	14.44	15.63	18.95	25.45	28.08	32.94	38.79	42.10	40.23	41.03	44.79	46.05	49.82	46.57
% Change from Control	0.15	0.10	-0.41	-0.90	-1.27	-0.73	-0.49	-0.65	-1.78	-0.45	-0.50	-0.19	-0.54	-0.85
Data are expressed as Mean ± S.D, Number of animals =10 Values are not statistically significant from control (p<0.05) Type of analysis - One-way ANOVA at 0.05 level of significance; @ Kruskal-Wallis Test

 

Table 2 Summary of body weight (g) – female

DAY
GROUP/DOSE (mg/kg b.w.)	1	8	15	22	^@29	36	43	50	^@57	64	71	78	85	90
G1 Control	192.49	216.38	234.70	245.59	255.17	269.38	275.95	284.68	290.21	297.37	303.80	306.11	311.42	315.16
	±	±	±	±	±	±	±	±	±	±	±	±	±	±
	10.05	12.30	10.30	11.30	11.19	15.71	17.13	16.89	15.88	15.00	16.92	16.78	18.02	19.59
G2 Low dose 250	193.40	214.47	233.01	245.11	255.43	266.47	271.38	282.38	287.99	293.66	300.92	304.50	307.49	308.87
	±	±	±	±	±	±	±	±	±	±	±	±	±	±
	9.63	10.53	7.68	10.20	11.83	10.90	11.61	10.83	10.92	11.57	11.31	10.98	11.85	11.76
% Change from Control	0.47	-0.88	-0.72	-0.20	0.10	-1.08	-1.66	-0.81	-0.76	-1.25	-0.95	-0.53	-1.26	-2.00
G3 Intermediate dose 500	194.33	217.34	237.28	250.12	256.83	265.43	271.97	280.51	288.03	295.90	299.83	302.54	307.74	309.51
	±	±	±	±	±	±	±	±	±	±	±	±	±	±
	10.52	14.35	14.70	12.32	11.19	13.19	13.75	14.90	15.40	15.74	16.79	17.61	16.86	16.96
% Change from Control	0.96	0.44	1.10	1.84	0.65	-1.47	-1.44	-1.46	-0.75	-0.49	-1.31	-1.17	-1.18	-1.79
G4 High dose 1000	193.53	215.45	232.52	244.23	257.13	268.50	274.75	284.48	289.81	296.26	301.55	305.64	310.25	312.17
	±	±	±	±	±	±	±	±	±	±	±	±	±	±
	7.98	9.01	11.59	11.20	7.72	7.59	8.23	10.30	11.23	12.21	11.78	11.90	12.84	13.78
% Change from Control	0.54	-0.43	-0.93	-0.55	0.77	-0.33	-0.43	-0.07	-0.14	-0.37	-0.74	-0.15	-0.38	-0.95
Data are expressed as Mean ± S.D, Number of animals =10 Values are not statistically significant from control (p<0.05) Type of analysis - One-way ANOVA at 0.05 level of significance; @ Kruskal-Wallis Test

 

Table 3 Hormone analysis (day 91) – male

Group	^@T3 (ng/ml)	T4 (ng/ml)	TSH (µIU/ml)
G1 Control	0.27	29.29	1.26
	±	±	±
	0.06	3.02	0.26
G2 Low dose 250 mg/kg b.w.	0.44*	29.49	1.24
	±	±	±
	0.15	4.28	0.27
% Change from Control	62.96	0.68	-1.59
G3 Intermediate dose 500 mg/kg b.w.	0.36*	30.27	1.22
	±	±	±
	0.10	4.54	0.23
% Change from Control	33.33	3.35	-3.17
G4 High dose 1000 mg/kg b.w.	0.26	30.94	1.26
	±	±	±
	0.04	5.73	0.20
% Change from Control	-3.70	5.63	0.00
Values are expressed as mean ± S.D, Number of animals =10; *Values are statistically different from control (p<0.05) Type of Analysis: - One-way ANOVA at 0.05 level of significance; @ - Kruskal-Wallis Test

 

Table 4 Hormone analysis (day 91) – female

Group	T3 (ng/ml)	^@T4 (ng/ml)	^@TSH (µIU/ml)
G1 Control	0.32	28.46	1.04
	±	±	±
	0.04	3.26	0.38
G2 Low dose 250 mg/kg b.w.	0.33	29.49	1.16
	±	±	±
	0.04	3.85	0.54
% Change from Control	3.13	3.62	11.54
G3 Intermediate dose 500 mg/kg b.w.	0.33	27.73	1.01
	±	±	±
	0.06	4.93	0.44
% Change from Control	3.13	-2.57	-2.88
G4 High dose 1000 mg/kg b.w.	0.31	29.44	1.02
	±	±	±
	0.06	5.64	0.37
% Change from Control	-3.13	3.44	-1.92
Values are expressed as mean ± S.D, Number of animals =10; *Values are statistically different from control (p<0.05) Type of Analysis: - One-way ANOVA at 0.05 level of significance; @ - Kruskal-Wallis Test

 

Applicant's summary and conclusion

Conclusions:

No adverse effects of TDI-Urone 80 on mortality, clinical signs, body weight, food consumption, hematology, clinical chemistry, organ weights, or gross and histologic pathology were observed in a subchronic toxicity study (OECD TG 408, GLP) conducted on Wistar rats.
The NOAEL is 1000 mg/kg bw/day.

Executive summary:

In a subchronic toxicity study (OECD TG 408, GLP) TDI-Urone 80 in distilled water was administered to Wistar (Crl:WI) rats in groups of 10/sex/dose (main groups) and 5/sex/dose (recovery groups) by oral gavage at dose levels of 0, 250, 500, 1000 mg/kg bw/day for 90 consecutive days . Rats in recovery groups were observed for an additional 28 days to evaluate any possible reversible effects after withdrawal of the treatment.

Clinical observations and feed consumption measurement were performed daily; body weights were determined weekly. Ophthalmological examinations were performed on all animals of all groups before the start of the dosing for main groups at week 13 and also at week 17 for recovery groups. Estimation of clinical chemistry, hematology and urinalysis were conducted at the end of 90 days treatment and 28 days recovery period.

There were no test item related effects in mortality, clinical signs, body weight, food consumption, hematology, clinical chemistry, organ weights, or gross and histologic pathology. The NOAEL is 1000 mg/kg bw/day.

This subchronic toxicity study in the rats is acceptable and satisfies the guideline requirement for a subchronic oral study (OECD 408) in rats.