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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-11-13 to 2023-03-31

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 414 (Prenatal Developmental Toxicity Study)

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

SOURCE OF TEST MATERIAL
- Source: Alzchem Trostberg GmbH
- Batch number of test material: 022704
- Purity: 2,4-TDI Urone: 78.6 (% w/w), 2,6-TDI Urone: 20.8 (% w/w)
- Expiry Date: 2022-08-13
- Storage: tightly closed polyethylene container in a dry, cool, and well-ventilated place (18 - 28 °C).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Dose formulation was prepared by mixing the test item with distilled water at the desired concentrations. The required test item formulations were prepared approximately 30 minutes prior to the start of dosing of animals.

Test animals

Species:

rat

Strain:

Wistar

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
- Age at study initiation: 12 - 13 weeks (at the start of acclimatization)
- Weight at study initiation: Males : 307.87 – 417.35 grams; Females : 217.75 – 282.86 grams (batch – 1) 207.66 – 295.12 grams (batch – 2)
- Fasting period before study: no
- Housing: clean, sterilized solid floored standard polypropylene rat cages covered with stainless steel grill with provisions for feed and water. Cages were placed on stainless steel racks. Cage rotation was done at weekly intervals. Clean and sterilized corn cob was used as bedding material. During acclimatization period, animals were housed in groups of two/three per
cage. After the acclimatization period, one female was housed with one male until the confirmation of mating (for a maximum period of 14 days) in cages equipped with stainless steel bottom grills with blotting paper underneath. After the confirmation of mating, the females were housed individually. Clean and sterile nesting materials (Nestlets-Sterilised Cotton Nesting Material Pad; Gusmer Enterprises, Inc; Lot no. 0224161000) was provided to all dams from gestation day (GD) 14 until scheduled sacrifice on GD 20. Male animals housed individually during the mating period, and females housed individually during the gestation period were provided with the enrichment devices (tunnels). Female animals for the study were allocated in two batches (each batch of 50 females). Phytoestrogen levels in bedding material was tested and was ensured to be within the acceptable limits (350 μg of genistein equivalents/gram of rodent laboratory feed or bedding material).
- Diet: ad libitum, laboratory rodent pellet feed (manufactured by Special Diet Services - England, supplied by Vivo Biotech Ltd., Hyderabad, India) Phytoestrogen levels in the feed was tested and was ensured to be within the acceptable limits (350 μg of genistein equivalents/gram of rodent laboratory feed or bedding material).
- Water: ad libitum, potable well water (IIBAT) processed through reverse osmosis
- Acclimation period:5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 – 22.9
- Humidity (%): 47 - 60
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: Batch - 1 Females: From: 2021-11-26 To: 2021-12-11 until 2021-12-21;
Batch - 2 Females: From: 2021-11-26 To: 2021-12-11 until 2021-12-31

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulation was prepared by mixing the test item with distilled water at the desired concentrations. The required test item formulations were prepared approximately 30 minutes prior to the start of dosing of animals. The test item was administered at approximately the same time (±1 h) each day as a single dose orally by gavage using a stainless steel oral intubation needle fitted to a graduated syringe. The dose volume was maintained at 1 ml/100 g body weight (b.w.). Animals in all the groups were administered the same volume of either the test item formulation or vehicle. Vehicle control group received the vehicle in the highest amount used as in the low dose group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulation was subjected to homogeneity, stability and active ingredient analysis once prior to the initiation of dosing and once during the second week of the dosing period. The sampling was performed from the top, middle, and bottom of the container with dose formulations in duplicate preparations for three dose levels. The analysis was performed by validated analytical method (IIBAT study no. 21196) at the Analytical Chemistry Department of IIBAT. The mean recovery for the dose verification samples analyzed prior to the initiation of dosing (November 17, 2022) ranged between 93.93 and 106.59%, and the mean recovery for the dose verification samples analyzed during the second week of dosing period (December 09, 2022) ranged between 97.08 and 105.10%.

Details on mating procedure:

- If cohoused: 50 male and 50 female animals were randomly cohabited
- M/F ratio per cage: 1:1 ratio (male: female).
- Length of cohabitation: 7 days if vaginal plug or sperm is not observed.
- After .7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 (Gestation Day (GD) 0) of pregnancy

Duration of treatment / exposure:

from implantation (GD 5) to one day prior to expected parturition (GD19).

Frequency of treatment:

daily

Duration of test:

27-39 days

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:

25 females/ test item treatment groups and 24 females for control group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses for the main study were selected based on the results of the sponsor study (Study no. 41104304).
- Time of day for (rat) dam blood sampling: gestation day (GD) 20

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily
- Animals were observed for pertinent behavioral changes, and all signs of toxicity, including mortality, morbidity to recorded in terms of time of onset, degree and duration. During pregnancy, maternal animals were monitored for signs of abortion or premature delivery, if any.

BODY WEIGHT: Yes
- Time schedule for examinations: females were weighed on GD 0, 3, 5, 8, 11, 14, 17, and on GD 20 prior to terminal sacrifice. During mating period, body weight of the females was taken at weekly intervals.

FOOD CONSUMPTION: Yes
The feed consumption of the animals during the main study was calculated for gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20. The feed consumption was calculated from the feed input and leftover feed data and reported as g/rat/day. The feed consumption was recorded during the mating period.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: GD 20
- Gross pathology: detailed examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. Evaluation of the dams during caesarean section and subsequent fetal analyses and sample collection were done randomly across available groups to avoid sampling bias.
- Organs examined: Thyroid gland (weight and histopathology)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- Serum: Yes
- Blood samples was collected individually from all dams on gestation day 20 and all non-pregnant females at scheduled termination day (GD 20). The blood samples were drawn from the animals through cardiac puncture at necropsy before dissecting the animals, in vials free of anticoagulant.
Blood samples were analyzed for thyroid hormones triiodothyronine (T3), thyroxine (T4) and
thyroid stimulating hormone (TSH) using ELISA method

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [one half per litter]
- Skeletal examinations: Yes: [one half per litter]
- Head examinations: Yes: [half per litter]
- Anogenital distance of all live rodent pups: yes

Statistics:

Data from non-gravid females was excluded from calculation of means and comparative statistics.

Continuous data variables (maternal body weights, gravid uterine weights, weight changes, feed consumption data, thyroid hormone data, fetal body weight (separately by sex, and combined), organ weights (absolute and relative), anogenital distance, and crown-to-rump length was analyzed for normality using Shapiro-Wilk test, and homogeneity of variance between groups was checked by Levene’s test. Where significant homogeneity is detected, a one-way analysis of variance (ANOVA) was carried out followed by parametric multiple comparison procedures using Dunnett's test for post hoc comparison. For non-normally distributed data, non-parametric ANOVA, Kruskal-Wallis test was used. If the results of the test are significant (p<0.05), the Mann-Whitney U test or Dunn’s test (pairwise comparison) was performed to identify significant group differences from control.
The group mean numbers of corpora lutea, implantation sites, viable fetuses, the mean litter proportions of prenatal data (% per litter of viable and nonviable fetuses, early/late resorptions, total resorptions, pre-/post-implantation loss and fetal sex distribution) was analysed using Kruskal-Wallis, non-parametric ANOVA to determine intergroup differences. If the results of the non-parametric ANOVA are significant (p<0.05), the Mann-Whitney-U test was performed to identify significant group differences from control.

The mean litter proportion (% per litter) of total fetal malformations and developmental variations (external, visceral, skeletal and combined) and of each particular external, visceral and skeletal malformation or variation was tabulated. The mean litter proportions of fetal malformations and
developmental variations was subjected to the Kruskal-Wallis non-parametric ANOVA test
followed by Mann-Whitney U test (if appropriate) as described above.
A p-value of <0.05 was considered statistically significant.

Indices:

Female Mating Index (%) = [(No. of vaginal plug or sperm positive females)/(Total no. of females cohabited with males)] x 100

Female Fertility Index (%) = [(No. of pregnant females)/(No. vaginal plug or sperm positive females)] x 100

Preimplantation loss (%) = [(number of corpora lutea – number of implantations)/(number of corpora lutea)] x 100

Postimplantation loss (%) = [(number of implantations – number of live fetuses)/(number of implantations)] x 100

Fetal death = resorptions + dead fetuses

AGD Index = AGD of fetus/Cube root of body weight

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity or behavioral changes was observed in any of the dams of treatment group or control groups throughout the experimental period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality/morbidity was observed in any of the animals of treatment or control group throughout the experimental period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item related effects on body weight and body weight change observed during gestation.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Feed consumption measured at various intervals during the gestation period (GDs 0-3, 3-5, 5-8, 8- 11, 11-14, 14-17 and 17-20) was comparable between the control and treatment groups, except there was a statistically significant decrease in the feed consumption in all the treatment groups of 250, 500, and 1000 mg/kg b.w. during GD 5-8. The decrease in the feed consumption was 8.48, 7.20 and 6.77% in the animals of 250, 500, and 1000 mg/kg b.w. respectively when compared with the control group. This decrease in the feed consumption was not having any correlation with the body weight of the animals as there were no changes in the body weight of treatment group animals during GD5 or GD8 when compared with the control group. Hence these changes in the feed consumption have no toxicological significance. The possible reason for the decrease in the feed consumption in the treatment group dams during GD 5-8 could be the animals adapting to the test item treatment during the initial days of dosing (dosing was initiated on GD5), which is evidenced by no change in the feed consumption data in the treatment group animals compared to the control animals for the subsequent gestation period (from GD 8 till GD 20).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormone levels (T3, T4, TSH) on GD 20 were comparable between control and treatment groups, except there was a statistically significant increase in T3 (0.24 ± 0.05 ng/mL) and T4 (30.74 ± 4.93 ng/mL) in 1000 mg/kg b.w. group when compared with control group. The increase in T3 and T4 parameters in 1000 mg/kg b.w. animals was 27.58 and 16.23% respectively when compared with control. The absolute thyroid weight was comparable between the control and the treatment groups and there was no statistically significant effect observed between the control (0.0295 ±0.0065 g) and high dose (0.0260 ±0.0059 g) and no test item related histopathological alterations in the thyroid.
Based on the missing correlates in organ weight and histopathology, this finding was considered as incidental and of no toxicological significance (for more information, please refer to “Details on results”).

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No clinical signs of toxicity or behavioral changes was observed in any of the dams of treatment group or control groups throughout the experimental period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The absolute and relative thyroid weight was comparable between the control and treatment groups, except there was a statistically significant decrease in the absolute (14.20%) and relative (11.97%) thyroid weight of 250 mg/kg b.w. animals, and in the relative (12.62%) thyroid weight of 1000 mg/kg b.w animals. These changes in the thyroid weight were not dose dependent as there were no changes in the absolute and relative thyroid weight in the 500 mg/kg b.w. animals, and no changes were observed in the absolute value in the 1000 mg/kg b.w. animals. Hence these changes were considered incidental and of no toxicological significance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Detailed examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents had revealed no abnormalities. No gross lesions were observed in the uterus and endometrial area between each implants.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item related histopathological findings were observed in the thyroid of treatment group of animals.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No test item related histopathological findings were observed in the thyroid of treatment group of animals.

Details on results:

Thyroid hormone results:
Analysis of literature had revealed that T3 and T4 values in GD 20 Wistar rats were ranging up to 3.92 ± 1.23 and 72.80 ± 30.04 ng/mL respectively (Deshmukh et al., 2021). GD 20 Long Evans rats had T3 and T4 values ranging up to 0.5366 ± 0.0543 ng/mL and 17.33 ± 1.96 ng/mL respectively (O’Shaughnessy et al., 2018). Moreover, in this study, there is no difference in the absolute thyroid weight of 1000 mg/kg b.w. animals and there is no test item related histopathological alterations in the thyroid. It is also evident that a statistically significant change in the thyroid hormone levels (T4) is common due to slight disturbances of the normal homeostasis that leads to hormonal fluctuations and are often not biologically relevant (Beekhuijzen et al., 2019). A prenatal developmental toxicity study in Wistar rats (study no. 21140) run in parallel to this study at IIBAT had the T3 and T4 values in the control animals ranging up to 0.58 ± 0.28 ng/mL (n=21) and 52.65 ± 4.54 ng/mL (n=21) respectively. Hence the difference in T3 and T4 values in the animals of 1000 mg/kg b.w. when compared with the control animals could be considered as incidental and is of no toxicological significance

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

None of the dams showed any signs of abortion or premature delivery prior to the scheduled termination on GD 20.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean number of corpora lutea, implantations sites, and mean percent pre-and postimplantation losses were similar between control and treatment groups (for more information, please refer to Table 1 in section "Any other information on results incl. tables").

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

No late resorptions or dead fetuses were observed in any of the dams of treatment and control groups. Hence, the fetal death was accounted only by the early resorptions in the dams (Fetal death = resorptions + dead fetuses). The mean number of early resorptions, mean number of live fetuses and the percent viable fetus per litter were similar between treatment and control groups (for more information, please refer to Table 1 in section "Any other information on results incl. tables").

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no dead fetuses observed in any treatment group or control group (for more information, please refer to Table 1 in section "Any other information on results incl. tables").

Changes in pregnancy duration:

not specified

Description (incidence and severity):

No premature delivery was observed in the dams

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Out of 99 animals which showed the evidence of copulation, 96 were found to be pregnant, and 3 were non-pregnant (one in control group and two in 250 mg/kg b.w.).

Other effects:

no effects observed

Description (incidence and severity):

The mean gravid uterine weight of treatment and control groups were similar

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean values of fetal weight (either separated by sex, or combined) were similar between treatment and control groups (for more information, please refer to Tables 2 and 3 in section "Any other information on results incl. tables").

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The fetal sex was found to be evenly distributed among the control and treatment groups.

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The mean values of anogenital distance and anogenital index were similar between treatment and control groups (for more information, please refer to Tables 2 and 3 in section "Any other information on results incl. tables").

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

Gross external examination of the fetus had revealed the following findings: subcutaneous hemorrhage in forelimbs of one female fetus (control; dam no. 2259, uterine position no. L3), blood clot above the left forelimbs of one female fetus (250 mg/kg b.w.; dam no. 2255, uterine position no. L2), subcutaneous hemorrhage in left pelvic region of one female fetus (500 mg/kg b.w; dam no. 2335, uterine position no. L2), and cleft palate with tongue protrusion in one male fetus (250 mg/kg b.w.; dam no. 2347, uterine position no. L6). All these findings were incidental and of no toxicological significance.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No test item related effects on the incidence of major or minor skeletal malformations were observed in the fetuses of treated or control dams. Cleft palate, which is classified as major malformation, was observed in one fetus of a dam belonging to the 250 mg/kg b.w. group, and wavy ribs, which is classified as minor malformation was observed in one fetus of the dam belonging to 1000 mg/kg b.w. (for more information, please refer to section “Details on embryotoxic/teratogenic effects”).

Visceral malformations:

no effects observed

Description (incidence and severity):

No remarkable soft tissue alterations were observed in any of the fetuses of the treated dams or control dams.

Details on embryotoxic / teratogenic effects:

Skeletal malformations/variations:
Overall incidence of these major and minor malformations occurring as single observations among the fetuses of their respective groups was very low, with 0.66% for cleft palate in the 250 mg/kg b.w. group and 0.58% for the occurrence of wavy ribs in the 1000 mg/kg b.w. group, respectively. Moreover, these incidences were compared with the literature (Spontaneous Malformations in Laboratory Animals: Frequency of External, Internal and Skeletal Malformations in Rats (Mortial,1987; Cong. Anom., 27: 147-206, 1987). As a result, these malformations were considered spontaneous and not treatment related. There are normal variants observed in the skeletal examinations of the fetuses of the treatment and control groups. These normal variants were spontaneous and not due to test item treatment and had no toxicological significance. The incidence of malformations (major/minor) and normal variants in the fetuses were statistically similar between treatment and control groups.

Effect levels (fetuses)

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

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table 1: Summary of animal uterine content data

Group No.	No. of. Corpora lutea	No. of. Implantation sites	Pre-implantation loss (%)	No. of. Early resorption	No. of. Late resorption	Total resorptions	No. of. Live fetus	No. of. Dead fetus	Total no. of fetus	Fetal death	Percent viable fetus per litter	Percent non-viable fetus per litter	Post-implantation loss (%)	^#Gravid uterine weight (g)
Group: G1 (Vehicle Control)	14.70	14.70	0.00	0.91	0.00	0.91	13.78	0.00	13.78	0.91	100.00	0.00	6.47	82.15
	±	±	±	±	±	±	±	±	±	±	±	±	±	±
	1.61	1.61	0.00	1.28	0.00	1.28	2.28	0.00	2.28	1.28	0.00	0.00	9.09	20.59
Group: G2 (250 mg/kg b.w)	14.78	14.78	0.00	1.48	0.00	1.48	13.30	0.00	13.30	1.48	100.00	0.00	9.60	81.07
	±	±	±	±	±	±	±	±	±	±	±	±	±	±
	2.41	2.41	0.00	1.62	0.00	1.62	2.36	0.00	2.36	1.62	0.00	0.00	9.53	12.80
% Change from Control	0.59	0.59	-	61.90	-	61.90	-3.47	-	-3.47	61.90	0.00	-	48.47	-1.31
Group: G3 (500 mg/kg b.w)	14.48	14.48	0.00	1.80	0.00	1.80	12.68	0.00	12.68	1.80	100.00	0.00	11.96	80.00
	±	±	±	±	±	±	±	±	±	±	±	±	±	±
	2.55	2.55	0.00	2.40	0.00	2.40	3.04	0.00	3.04	2.40	0.00	0.00	15.44	16.93
% Change from Control	-1.47	-1.47	-	97.14	-	97.14	-8.00	-	-8.00	97.14	0.00	-	84.99	-2.62
Group: G4 (1000 mg/kg b.w)	14.88	14.88	0.00	1.24	0.00	1.24	13.64	0.00	13.64	1.24	100.00	0.00	8.36	86.32
	±	±	±	±	±	±	±	±	±	±	±	±	±	±
	1.83	1.83	0.00	2.22	0.00	2.22	2.83	0.00	2.83	2.22	0.00	0.00	14.94	16.39
% Change from Control	1.25	1.25	-	35.81	-	35.81	-1.03	-	-1.03	35.81	0.00	-	29.23	5.07
Data from the non-pregnant animal(s) is excluded from the calculation of mean and SD and from the statistical analysis; n=23 for G1 and G2; n=25 for G3 and G4; Values are not statistically different from control (P > 0.05) Type of analysis - Kruskal Wallis at 0.05 level of significance

 

Table 2 Summary of fetal data (male)

 

Group/Dose	AGD (mm)	AGD Index	Fetal weight (g)	Crown Rump (mm)
G1 (Vehicle Control)	2.29	1.43	4.0703	37.32
	±	±	±	±
	0.72	0.45	0.3956	2.81
G2 (Low dose) - 250 mg/kg b.w.	2.18	1.37	3.9543	36.88
	±	±	±	±
	0.73	0.45	0.4642	3.28
% Change from Control	-4.66	-3.92	-2.85	-1.16
G3 (Intermediate dose) - 500 mg/kg b.w.	2.09	1.31	4.0044	37.14
	±	±	±	±
	0.70	0.43	0.5083	2.78
% Change from Control	-8.79	-8.30	-1.62	-0.47
G4 (High dose) - 1000 mg/kg b.w.	2.09	1.31	4.0321	37.28
	±	±	±	±
	0.71	0.43	0.5021	2.80
% Change from Control	-8.59	-8.33	-0.94	-0.10
Data are expressed as Mean ± S.D Values are not statistically significant (P>0.05) Kruskal Wallis at 0.05 level of significance

 

Table 3 Summary of fetal data (female)

Group/Dose	AGD (mm)	AGD Index	Fetal weight (g)	Crown Rump (mm)
G1 (Vehicle Control)	2.11	1.33	3.9932	37.25
	±	±	±	±
	0.74	0.45	0.4179	2.58
G2 (Low dose) - 250 mg/kg b.w.	2.18	1.38	3.9669	36.99
	±	±	±	±
	0.73	0.45	0.4197	2.66
% Change from Control	3.39	3.66	-0.66	-0.69
G3 (Intermediate dose) - 500 mg/kg b.w.	2.08	1.31	4.0032	37.13
	±	±	±	±
	0.70	0.43	0.5086	2.78
% Change from Control	-1.26	-1.22	0.25	-0.31
G4 (High dose) - 1000 mg/kg b.w.	2.09	1.31	4.0322	37.28
	±	±	±	±
	0.71	0.43	0.5014	2.80
% Change from Control	-1.12	-1.33	0.98	0.09
Data are expressed as Mean ± S.D Values are not statistically significant (P>0.05) Kruskal Wallis at 0.05 level of significance

 

Applicant's summary and conclusion

Conclusions:

In a developmental toxicity study (OECD 414, GLP) conducted in rats, TDI-Uron 80 did not cause maternal or developmental toxicity up to the maximum recommended dose of 1000 mg/kg b.w./day. The NOAEL for maternal and developmental effects for TDI-Urone 80 is greater than 1000 mg/kg b.w./day.

Executive summary:

In a developmental toxicity study (OECD 414, GLP) TDI-Urone 80 was administered in distilled water to 24 (control group) or 25 (test item treatment groups) female Wistar (Crl:WI) rats/dose by oral gavage at dose levels of 0, 250, 500, or 1000 mg/kg bw/day from days 5 through 19 of gestation.

There were no treatment-related effects in mortality, clinical signs, body weight or food consumption in maternal animals. Moreover, exposure to TDI-Urone 80 did not cause effects on gravid uterine weights, number of corpora lutea, number of implantations sites, and mean percent pre-and post-implantation losses. The mean number of early resorptions, mean number of live fetuses and the percent viable fetus per litter were similar between treatment and control groups. The maternal NOAEL is greater than 1000 mg/kg bw/day.

There were no treatment-related effects in developmental parameters. The mean values of fetal weight (either separated by sex, or combined), anogenital distance, anogenital index, and crown-to-rump length were similar between treatment and control groups. No effects of treatment with TDI-Urone 80 were observed upon visceral and skeletal fetal examinations. The developmental NOAEL is greater than 1000 mg/kg bw/day.

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rats.