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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

HAT ISO was tested for toxicity to reproduction in two one-generation studies in the rat. No effects on reproduction were found in either study, up to an exposure level of 1000 mg/kg bw/day (limit dose).

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Specific details on test material used for the study:

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

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;


:

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 ).

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

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).

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: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

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.

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

Key result

Critical effects observed:

no

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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.

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

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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.

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and guideline study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Two studies on reproduction were performed assessing the reproductive potential of HAT ISO. In the key study test item was administered up to the limit dose of 1000 mg/kg bw/day. The second study is considered a supporting study, as the top dose had to be reduced due to limited test item availability.

 

In the key study HAT ISO was tested according to EU Method B.34. The test item was administered in by oral gavage at a limit dose of 1000 mg/kg bw/day. HAT ISO showed no signs of maternal toxicity and also no signs of toxicity to the offspring and for both NOAEL values of 1000 mg/kg bw/day were revealed.

 

The supporting study tested HAT ISO according to OECD Guidelines 415/414. Due to limited availability of test material, the top dose had to be reduced to 600 mg/kg bw/day. Oral administration of HAT ISO in male and female rats at the dose levels of 150, 300 and 600 mg/kg bw/day did not induce toxicological effects in either parent animals or offspring. Thus, the NOAELs for parent animals and offspring were 600 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

HAT ISO was tested for developmental toxicity/teratology in a study in rabbits according to OECD Guideline 414 and OECD Guidance no. 43 on Mammalian Reproductive Toxicity Testing and Assessment (24 July 2008). HAT ISO showed no maternal toxicity, no developmental toxicity and no teratogenicity by administration of the test item by oral gavage in doses up to 1000 mg/kg bw/day (limit dose).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-04-01 to 2009-05-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 January 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guidance no. 43 on Mammalian Reproductive Toxicity Testing and Assessment; 24th July 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

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

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sándor Ferenc (formerly TETRABBIT Kft.) 2173 Kartal, Császár út 135, Hungary;
- Age at study initiation: young adult rabbits; approximately 4 month;
- Weight at study initiation: mean weight 3547 - 4227 g;
- Fasting period before study: Not stated;
- Housing: individually in AAALAC approved metal wire rabbit cages;
- Diet: PURINA Base - Lap gr. diet for rabbits; Supplier: AGRIBRANDS Europe Hungary PLC, H-5300 Karcag, Madarasi
- Water: Tap water for human consumption; via automatic water system ad libitum;
- Acclimation period: at least 5 days;


ENVIRONMENTAL CONDITIONS
- Temperature: 17.3 - 21.65 °C;
- Humidity: 32 - 98.79 % R.H.
- Air changes: not stated;
- Photoperiod: 12 hours light/12 hours dark; from 6:00 a.m. to 6:00 p.m.;

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test item was administered to pregnant female rabbits daily by oral gavage on a 7 days/week basis, at similar times in the morning, from GD 6 to GD 27 (one day prior to caesarean section and necropsy examination). Prior to the insemination procedure, the animals were acclimatised for at least 5 days (see also dosing scheme). Control animals were treated concurrently with the vehicle only.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verification was made by HPLC Analysis and detection on a DAD-Detector at 250 nm wavelength;

Details on mating procedure:

The female rabbits were artificially inseminated. The day of insemination was regarded as Day 0 of gestation (GD 0). Synchronisation of the oestrus cycle of the does was performed 48 hours prior to insemination by administration of PMSG (gonadotropin) hormone (40 IU/female, sc). The insemination procedure was performed by the breeder at LAB Research Ltd. in batches (staggered; see detailed schedule in Appendix 7). Each female was inseminated with diluted sperm containing at least 2 million spermatozoa. Simultaneously with the artificial insemination, the does’ ovulation was provoked with 1 ml buserelin-based compound (0.2 mL/animal, i.m.) (see Details of Other Materials section). The sperm originated from New Zealand White male rabbits from the same source as the females. Females were randomly assigned to dose groups on the basis of their body weight on the day of insemination.

Duration of treatment / exposure:

The test item was administered to pregnant female rabbits daily by oral gavage on a 7 days/week basis, at similar times in the morning, from GD 6 to GD 27 (one day prior to caesarean section and necropsy examination). Prior to the insemination procedure, the animals were acclimatised for at least 5 days (see also dosing scheme). Control animals were treated concurrently with the vehicle only.

Frequency of treatment:

The test item was administered to pregnant female rabbits daily by oral gavage on a 7 days/week basis, at similar times in the morning, from GD 6 to GD 27 (one day prior to caesarean section and necropsy examination).

Duration of test:

The test item was administered to pregnant female rabbits daily by oral gavage on a 7 days/week basis, at similar times in the morning, from GD 6 to GD 27

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

350 mg/kg bw/day (actual dose received)

Dose / conc.:

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

No. of animals per sex per dose:

24 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The test item was administered to pregnant female rabbits daily by oral gavage on a 7 days/week basis, at similar times in the morning, from GD 6 to GD 27 (one day prior to caesarean section and necropsy examination). Prior to the insemination procedure, the animals were acclimatised for at least 5 days (see also dosing scheme). Control animals were treated concurrently with the vehicle only.

Maternal examinations:

Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).

Ovaries and uterine content:

The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix were weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses. Where no implantation sites were evident but corpora lutea were present, the uterus was
stretched and held in front of a light source to identify properly the implantation sites. Uteri that appeared non-gravid were further examined to confirm the non-pregnant status by Salewski staining. The corrected body weight was calculated (body weight on gestation day 28 – weight of the gravid uterus).

Fetal examinations:

The live foetuses were identified, weighed individually, and the litter mean was calculated. The crown-rump length of foetuses was measured (accuracy 1 mm) and the litter mean was calculated. All foetuses were externally and viscerally examined, and sex determined. The heads from approximately half of each litter were removed and processed for evaluation of soft tissue alterations (including eyes, brain, nasal passages and tongue), using fixation in Sanomiya mixture for Wilson-sections. After fixation the heads were examined by Wilson's free-hand razor blade method. Then all foetuses were prepared for skeletal examination. The skeletons were examined after double staining with acetic alcian blue + KOH-alizarin red S staining (cartilage and bone staining). All abnormalities (external, soft tissue and skeletal malformations, and variations) found during the foetal examinations were recorded.

Statistics:

The statistical evaluation of data was performed with the program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett`s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was made. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result, inter-group comparisons were performed using Kruskal-Wallis and Mann-Whitney U-test.

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):

no effects observed

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

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects: no effects observed

Details on maternal toxic effects:
There were no test-item related clinical signs noted following administration of HAT-ISO to pregnant does from GD 6 to 27, by daily oral gavage, at dose levels up to and including 1000 mg/kg bw/day HAT-ISO, at a dose volume of 2 mL/kg bw.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: no effects observed at highest dose tested

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no effects observed

Details on embryotoxic / teratogenic effects:
No test-item related effects were noted in the number of dead/viable foetuses, or their sex distribution. The sex ratios were similar in the control and treated groups. The litter mean values of foetal weight and crown-rump length were similar in the foetuses from the treated and control does. There were no abnormalities considered toxicologically significant and/or possibly ascribed to HAT-ISO administration at external, visceral or skeletal foetal examinations.

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: no effects observed at highest dose tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

No mortality or test-item related clinical signs occurred during the study.

HAT-ISO administered to artificially-inseminated New Zealand White female rabbits by oral gavage at dose levels of 100, 350, and 1000 mg/kg bw/day in corn oil from GD 6 to 27, led to dose and time-dependent increases in does’ food consumption, correlated with higher body weights and/or body weight gain at 1000 mg/kg bw/day. In the absence of any maternal or developmental toxicity, these changes were not considered an adverse effect of the test item in the conditions of this study.

There were no test item related effects on any of the maternal reproductive parameters examined during the study. The number of corpora lutea and implantation sites was similar in the treated groups compared to control animals. There were no differences between the treated and control animals regarding the pre-implantation loss, early and late embryonic death, postimplantation loss, or total intrauterine mortality; the mean values and/or percentile were comparable, or lower in the HAT-ISO treated animals, with no statistically significant variations compared to the animals administered vehicle control (corn oil) only. No macroscopic findings or treatment-related changes in the gravid uterine weight were recorded in the treated pregnant does surviving to termination when compared to the controls. The placenta was normal in all the does examined.

As necropsy followed by histopathology evaluation of the females with macroscopic findings, no macroscopic or microscopic findings recorded were ascribed to HAT-ISO administration. One low dose doe administered 100 mg/kg bw/day (1/24, female 2523) aborted and was necropsied on GD27; based on the isolated incidence and in the absence of similar findings in this group, or at higher dose levels, abortion in this animal was considered spontaneous and not related to treatment.

No test-item related effects were noted in the number of dead/viable foetuses, or their sex distribution. The sex ratios were similar in the control and treated groups. The litter mean values of foetal weight and crown-rump length were similar in the foetuses from the treated and control does. There were no abnormalities considered toxicologically significant and/or possibly ascribed to HAT-ISO administration at external, visceral or skeletal foetal examinations.

In conclusion, under the conditions of this study, the no-observed-effect-level values are:

NOAEL maternal toxicity: 1000 mg/kg bw/day

NOAEL developmental toxicity: 1000 mg/kg bw/day

NOEL teratogenicity: 1000 mg/kg bw/day

Conclusions:

HAT-ISO was tested for Developmental Toxicity / Teratology in rabbits by oral gavage during the gestation period. No mortality or test-item related clinical signs occurred during the study. The no-observed effect levels are:
NOAEL maternal toxicity: 1000 mg/kg bw/day;
NOAEL developmental toxicity: 1000 mg/kg bw/day;
NOEL teratogenicity: 1000 mg/kg bw/day.
No classification and labelling is necessary according to Regulation 1271/2008/EEC (CLP).

Executive summary:

A Prenatal Developmental Toxicity/Teratology Study was performed in the rabbit according to OECD 414 and the draft OECD guidance document 43. The objective of this study was to assess the effects of HAT-ISO on pregnant females and developing conceptuses when administered to artificially inseminated, assumed pregnant New Zealand White rabbits. Test substance was administered daily by oral gavage at dose levels of 100, 350, and 1000 mg/kg bw/day.

No mortality or test-item related clinical signs occurred during the study.

Under the conditions of this study, the no-observed-effect-level values are:

NOAEL maternal toxicity: 1000 mg/kg bw/day;

NOAEL developmental toxicity: 1000 mg/kg bw/day;

NOEL teratogenicity: 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

GLP and guideline study

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The testing of HAT ISO for prenatal developmental toxicity/teratogenicity was performed in a study in rabbits according to OECD 414 and draft OECD Guidance Document 43. Test substance was administered daily by oral gavage at dose levels of 100, 350, and 1000 mg/kg bw/day. No mortality or test-item related clinical signs were observed in parent or F1 offspring animals during the study. Thus, maternal and offspring NOAELs were 1000 mg/kg bw/day (limit dose). The teratotoxicity NOEL was 1000 mg/kg bw/day.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

HAT ISO was tested in two one-generation reproduction toxicity studies in rats according to EU Method B.34 / OECD Guideline 415. No reproduction toxicity effects were observed following oral administration of the test item up to a dose of 1000 mg/kg bw/day (limit dose). The NOAELs for parent animals and offspring were determined to be 1000 mg/kg bw/day.

Testing of prenatal developmental toxicity / teratogenicity of HAT ISO was performed in rabbits according to OECD 414/OECD Draft Guidance Document no. 43. The NOAELs for parent animals and offspring were determined to be 1000 mg/kg bw/day (limit dose).

Based on results obtained for reproductive and developmental toxicity, HAT ISO does not require classification, according to Regulation 1272/2008/EC (CLP) as amended for the tenth time.

Remark on ECHA common screening round 3 (Janaury 2016):

Based on information by ECHA from 26 January 2016 the substance was selected based on an automated screening for potential further evaluation. The endpoint of concern was toxicity to reproduction. The substance potentially contains a harmonised reproductive toxicant (category 1A/1B) above the generic or specific concentration limits in at least one registration. The impurity of concern is N,N'-dimethylformamide which has a harmonized classification for toxicity to reproduction cat. 1B (H360D). This impurity is no longer present in the registered substance. Please refer to IUCLID section 1.2 for an updated substance ID and a justification for update of this section. Nevertheless, the above mentioned studies were conducted using the old batch potentially containing very low amounts of N,N'-dimethylformamide (below 0.1%, which is below the generic concentration limit for classification). Based on the presented results, the substance showed absolutely no adverse effects on fertility and development. Potential adverse effects caused by the impurity N,N'-dimethylformamide are thus covered by these studies. In conclusion the registered substance is judged to have no potential for toxicity to reproduction and further evaluation of this endpoint by a Member State Competent Authority or ECHA is not required.