1,1'-(methylenedi-4,1-phenylene)bis(3-butylurea)

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-06-13 to 2009-09-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.34 (One-Generation Reproduction Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

416-600-4

EC Name:

-

Cas Number:

77703-56-1

Molecular formula:

C23H32N4O2

IUPAC Name:

3-butyl-1-[4-({4-[(butylcarbamoyl)amino]phenyl}methyl)phenyl]urea

Specific details on test material used for the study:

Analytical purity: > 99.4 %
Lot/batch No.: HAT-ISO 05-12-06

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories; 69592 L'Arbresle cedex; France
- Age (on receipt): Males: about 5 weeks old; females: about 8 weeks;
- Weight at study initiation: at treatment initiation (D 1): males ranging from 276.6 to 338.3 g; females ranging from 185.1 to 232.0 g;
- Fasting period before study: not indicated;
- Housing: males at a rate of 3 per cage, in BC3 polycarbonate cages with stainless steel lid; females were housed individually in same cage type;
- Diet: A04-10 pelleted rodent diet; supplier: SAFE; 89290 Augy; France; ad libitum;
- Water: acidified (pH = 2.5) tap water ad libitum; from polypropylene bottles;
- Acclimation period: 5 days before beginning of treatment;


ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2 °C;
- Humidity: 50 +/- 20 % R.H.;
- Air changes: 10 cycles per hour;
- Photoperiod: 12 hours dark/12 hours light;


:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

The test preparation was administered by oral route (gavage) at a single dose level of 1000 mg/kg bw/day (limit test). The dosing volume was kept constant at 10 mL/kg bw. The dose level of 1000 mg/kg was chosen since the test element is of low toxicity as demonstrated in repeated-dose studies. Concurrently to the treated group (group 2), the animals from the control group (group 1) were handled similarly to the treated group animals receiving the control element (vehicle: 1 % CMC hydrogel) in the same volume (10 mL/kg bw). The test preparation was administered by oral route to all animals of the P generation group 2, each day approximately at the same time of day (between 9:00 and 12:00 a.m.) during 10 weeks (males) and 2 weeks (females) before mating and during the mating period (3 weeks maximum).

Details on mating procedure:

A 1 : 1 (1 male to 1 female) mating was performed. The male was placed with the same female each morning till pregnancy occurred (for a 3 weeks maximum period). After each mating session, the females were examined for presence of a vaginal plug. Day 0 of pregnancy (P0) was defined as the day a vaginal plug was found.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical method: HPLC with UV detection;
Appliance: VWR Hitachi L-2140;
Spectrometer: DAD-spectrometer VWR Hitachi L-2450;
Column: Phenomenex Gemini 3uO6 Phenyl 110A, 150 x 4.6 mm, 3 µ;
Pre-Column: Phenomenex C18, 2 x 4 mm;
Mobile Phase: A: Methanol/Water (10:50)(v/v); B: Acetonitrile
Start Eluent: 60 % A during 5 min;
Gradient: 3 % a / min to 100 % B;
Flow: 0.8 mL/min;
Detection: 250 nm;
Injection volume: 10 µL;
Total Runtime: 40 min;

Duration of treatment / exposure:

The test preparation was administered by oral route to all animals of the P generation group 2, each day approximately at the same time of day (between 9:00 and 12:00 a.m.) during 10 weeks (males) and 2 weeks (females) before mating and during the mating period (3 weeks maximum). In males, the daily dosing was continued till the treatment period was equal to 13 weeks. In females, the daily dosing was continued throughout the pregnancy and up to the weaning of the offspring (three weeks after parturition).

Frequency of treatment:

Each preparation was orally administered at one time, at a single dose, to each animal, by gavage using a syringe with appropriate volume (2.5 ml, 5 ml or 10 ml), fitted with a suitable sized stainless steel intubation’s cannula (76 mm x 15/10th).

Details on study schedule:

The animals were allocated into 2 groups of 48 animals:
- group 1 (control group) = 24 males and 24 females received 1% CMC hydrogel
- group 2 (treated group) = 24 males and 24 females received the test element in the vehicle
The test preparation was administered by oral route (gavage) at a single dose level of 1000 mg/kg bw/day (limit test). The dosing volume was kept constant at 10 ml/kg bw. The dose level of 1000 mg/kg was chosen since the test element is of low toxicity as demonstrated in repeated-dose studies (subacute toxicity study ref. 907/002 of 12/09/1994 PHARMAKON Europe and a one generation reproduction study with information about possible teratogenicity ref. Tf 742 / 99-3117 of September 13, 2000 performed in the Test Facility). Concurrently to the treated group (group 2), the animals from the control group (group 1) were handled similarly to the treated group animals receiving the control element (vehicle: 1 % CMC hydrogel) in the same volume (10 mL/kg bw ).

Doses / concentrations

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

The animals were allocated into 2 groups of 48 animals :
- group 1 (control group) = 24 males and 24 females received 1% CMC hydrogel
- group 2 (treated group) = 24 males and 24 females received the test element in the vehicle

Control animals:

yes, concurrent vehicle

Details on study design:

Control of the concentrations:
Concentration and homogeneity of test element preparations were determined on the 1st week of male rats’ treatment (Week 1), during males and females treatment before mating (Week 9), during the pregnancy (Week 14) and during nursing period (Week 17); after preparation of the test element, sampling of the dose preparation was performed at room temperature.

Analytical method: see above.

Positive control:

NA

Examinations

Parental animals: Observations and examinations:

Parental (P) animals were examined for mortality, clinical observations, body weight, food consumption, delivery, gross necropsy and histopathological parameters.

Postmortem examinations (parental animals):

Parental animals, if found dead during the experiment or sacrificed at the end of the experimental period (13 weeks for males ; on the 21st day p.p. for the females), would have been submitted to a macroscopic examination for any structural abnormalities or pathological changes with a particular attention paid to the organs of the reproductive system.

Postmortem examinations (offspring):

All the pups, if found dead during the lactation period (when not macerated), would have been examined macroscopically for possible defects and/or cause of death in this study.

Statistics:

For each generation, body weight changes were analyzed separately by a two-way analysis of variance for repeated measurements in time taking the "time" and "treatment” factors into consideration. Should a statistically significant dosing effect be found, the mean of the control group was compared with that of the treated group using the Fisher's test. Other numerical results will be analyzed separately, parameter by parameter. Once variance homogeneity between groups (Levene’s test) and data distribution normality (Shapiro-Wilk’s test) were analyzed the treated group was compared with the control group using the Student's test for unpaired series or a non- parametric test (Mann-Whitney’s U test).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Details on results (P0)

No mortality occurred during the experiment. No signs of toxicity were noted in the animals from the 2 groups during the experiment. Treatments had no effect on the appearance or behaviour of the parental animals. No signs of difficult or prolonged parturition were observed. At the end of the experimental period, no abnormalities were detected in the detailed clinical examination performed on control and treated males and females; in a series of functional observation battery tests (such as home cage observations, handling observations, open field observations, sensory observations, physiological observation and neuromuscular observations) no treatment-related functional abnormalities in either sex were found in this study.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at the limit dose tested

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Mean litter size and mean number of stillbirths and live births per litter were similar for the 2 experimental groups. The sex ratio was not affected by the test element. The examination of litters from Group 2 showed two small size newborns (runts) in female No 6180 (1/9 male) and in female No 6177 (1/6 female) and in one offspring from the female No 6188 the abdomen area was blackish coloured. No abnormalities were observed at the external examination of the two stillbirth animals noted in control group (F 6173) and treated group (F 6178). Under the experimental conditions adopted, no differences were evidenced between the pups from dams treated with the test element and those from the control dams. The physical and functional pup development was not altered by HAT-ISO treatment.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at the limit dose tested

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

HAT ISO was administered orally (gavage) at a single dose level of 1000 mg/kg bw/day (limit test) to one group of male and female rats. Limit test was chosen because of the low toxicity of HAT ISO.

 

A control animals' group (group 1) received orally the same volume (10 ml/kg bw) of vehicle (1% CMC hydrogel) under the same conditions.

The males were daily dosed during 13 weeks i.e. 10 weeks prior to mating, during mating period, at least 3 weeks after the beginning of mating period until then they were sacrificed.

The females were dosed daily during 2 weeks prior to mating (i.e. two complete oestrous cycles), during the mating period (3 weeks maximum), pregnancy and up to weaning of the offspring (3 weeks after parturition).

During the experimental period, males and females from the parental (P) generation were daily observed for possible signs of toxicity. Clinical signs, body weights and food consumption, were recorded.

After delivery, the pups were regularly observed till weaning (body weight, clinical signs, physical and functional development) and then were sacrificed.

Parental animals were sacrificed at the end of the experimental period (13 weeks for males ; on the 21^stday post partum for the females) and submitted to a complete macroscopic examination (gross necropsy, organ weight) and to histopathological examination.

Following organs were weighed and subjected to a full histopathological examination:

 - Males: testes, epididymides, seminal glands, prostate and coagulating gland, thymus, spleen, liver, mesenteric lymph nodes, brain, pituitary and thyroid

-  Females: ovaries, uterus with cervix and vagina

Results

Observation of Parental animals' (males and females)

No mortality or toxicity signs were noted during the course of the experiment.

HAT-ISO treatment did not affect body weight and food consumption. In females, HAT-ISO did not induce any undesired effects on pregnancy delivery and nursing period. The number of pregnant females in the two groups was similar (15/24 in HAT-ISO group (62.5%) versus 16/24 in control group (67%)).

Offspring examination

No significant differences were noted between pups from females of group receiving HAT-ISO and those receiving vehicle.

The parameters determined (pup number, sex ratio, litter weight and body weight of pups, clinical signs, physical and functional development, post natal losses) did not reveal difference between the two groups. The viability index from D4 post partum to weaning (D21 p.p.) was higher in HAT-ISO group (99%) than in control one (89%).

Post mortem examination

* Males sacrificed at 13 weeks

No macroscopic or microscopic findings, indicative of a toxic effect of HAT-ISO were noted.

A statistically significant decrease in absolute pituitary weight (-12.3%, p<0.05) was noted in HAT-ISO treated animals ; this change was considered to have no toxicological importance since it was not associated with any histopathological change in this organ.

* Females sacrificed on D21 p.p.

No macroscopic or microscopic findings, indicative of a toxic effect of HAT-ISO were noted.

Mean number of corpora lutea and of implantation sites determined in pregnant females were comparable in the two groups. Pre implantation losses were similar (19% in HAT-ISO group versus 18% in control group).

* Examination of pups

Macroscopic examination of dead pups during the lactation period did not reveal difference between the two groups.

Conclusion

Under the experimental conditions adopted, daily oral administration of HAT-ISO by gavage at the level of 1000 mg/kg bw/day showed no parental animal systemic toxicity and did not affect their reproductive performance compared to controls. HAT-ISO treatment had no effects on litter size, sex ratio, offspring viability indices and physical development of the F1 generation.

Given the absence of adverse effects on reproductive and developmental parameters, the no observed adverse effects level (NOAEL) for HAT-ISO was considered to be equal (or greater) to 1000 mg/kg bw/day in this study.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions adopted, daily oral administration of HAT-ISO by gavage at the level of 1000 mg/kg bw/day showed no parental animal systemic toxicity and did not affect their reproductive performance compared to controls. HAT-ISO treatment had no effects on litter size, sex ratio, offspring viability indices and physical development of the F1 generation.
Given the absence of adverse effects on reproductive and developmental parameters, the no observed adverse effects level (NOAEL) for HAT-ISO was considered to be equal (or greater) to 1000 mg/kg bw/day in this study.
No classification and labelling is necessary according to Regulation 1272/2008/EC (CLP).

Executive summary:

HAT ISO was tested for reproductive toxicity in a one-generation toxicity study in rats.

HAT ISO was administered orally (gavage) at a single dose level of 1000 mg/kg bw/day (limit dose) to one group of male and female rats. A control animals' group (group 1) received orally the same volume (10 mL/kg bw) of vehicle (1% CMC hydrogel) under the same conditions.

No mortality or toxicity signs were noted during the course of the experiment in parental animals (males and females). No significant differences were noted between pups from females of group receiving HAT-ISO and those receiving vehicle.

No macroscopic or microscopic findings, indicative of a toxic effect of HAT-ISO were noted. Given the absence of adverse effects on reproductive and developmental parameters, the no observed adverse effects level (NOAEL) for HAT-ISO was considered to be equal 1000 mg/kg bw/day (limit dose) for parent and offspring in this study.