6-methyl-2-oxoperhydropyrimidin-4-ylurea

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No data available. 

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP. Since this is a read-across from a structural analogue substance (CAS 6104-30-9), the reliability was set from RL1 to RL2.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: males: 8 weeks; females: 10 weeks
- Weight at study initiation: males: 276 g mean bw; females: 228 g mean bw
- Housing: housed individually in suspended wire-mesh cages, females shifted 4 days before lactation to polycarbonate cages in a barrier rodent unit.
- Diet: A04 C pelleted diet ad libitum, distributed weekly
- Water: tap water ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21± 2
- Humidity (%): 50±20
- Air changes (per hr): about 12 cycles of filtered, non recycled air.
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The vehicle was 0.5% aqueous carboxymethylcellulose solution prepared using:
- purified water, obtained by reverse osmosis using a Milli-Ro 8 plus apparatus (Millipore SA, Saint-Quentin en Yvelines, France).
- carboxymethylcellulose, batch No. 69H0028, supplied by Sigma (Saint-Quentin-Fallavier, France).

The test substance was administered as a suspension in the vehicle.
The test substance was ground using a mortar and pestle, suspended in the vehicle in order to achieve the concentrations of 10, 30 and 100 mg/mL
and then homogenized using a magnetic stirrer.
The test substance dosage forms were made daily (preparation stable for 3 hours) .

Details on mating procedure:

- M/F ratio per cage: 1 male/1 female of the same dose group
- Length of cohabitation: during the night
- Proof of pregnancy: [vaginal plug or sperm in vaginal smear] referred to as [day 0] of pregnancy
- Each female was placed with the same male until mating occured or 14 days had elapsed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the test substance preparations: concentration and homogeneity.
Three samples of each control and test substance dosage form (top, middle and bottom of the flasks ) prepared for use during the first week and the
last week of treatment were taken .
They were stored at -20°C pending dispatch, to the Sponsor, for analysis of concentration and homogeneity

Duration of treatment / exposure:

Exposure period: males: pre-mating, mating (14days) and post-mating, for a total of 34 days, females: pre-mating, mating, pregnancy and lactation until day 4 post partum
Premating exposure period (males): 15 days
Premating exposure period (females): 15 days
Duration of test: males: until sacrifice in the post-mating period, females: until day 4 post partum

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

no futher data

Remarks:

Doses / Concentrations:
100; 300; 1000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose-levels were specified by the Sponsor, following the results of a prev iously conducted prenatal developmental
toxicity study in Wistar rats (BASF Project No. 30R0727/90116). The oral route was selected since it is the route of exposure, which is requested by
regulatory authorities for this type of product.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily at least once

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first day of treatment and then once a week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: male animals: first day of treatment, then once a week until sacrifice; female animals: first day of treatment, then once a week until mated (or until sacrifice) and on days 0, 7, 14 and 20 post-coitum and days 1 and 4 post-partum

FOOD CONSUMPTION AND COMPOUND INTAKE :
The quantity of food consumed by each animal was recorded once a week, from the first day of each of the pre-mating, gestation and lactation periods. During the mating period, the food consumption was noted for neither males nor females .

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations: testis weight, epididymis weight, morphological examination of the testes: tailed and round spermatids, spermatocytes, spermatogonia, different stages of spermatogonic cycle

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
-The following parameters were examined in [F1] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
- yes, for external abnormalities; possible cause of death was not determined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after the completion of the treatment period: after the end of the mating period: on study day 35 (total treatment period was 34 days)
- Maternal animals: All surviving animals after the completion of the treatment period: the females and their pups were killed on day 5 post partum. Female animals showing no evidence of mating were killed 24-26 days after the last day of mating.

GROSS NECROPSY
- A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces , all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. In the parent females, the corpora lutea and implantation sites were recorded. In apparently non-pregnant or un-mated females, the presence of implantation scars on the uterus was checked using ammonium sulphide staining technique.

HISTOPATHOLOGY / ORGAN WEIGHTS
- body weight and organ weights were examined (adrenals, brain, heart, kidneys, liver, spleen, thymus; additionally in males: testes and epididymides; females:ovaries)
- Histopathology: macroscopic lesions, adrenals, brain, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes, ovaries, prostate, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, sternum, stomach, testes, thymus, thyroids and parathyroids, trachea, urinary bladder, uterus

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 5 days of age.
- These animals were subjected to postmortem examinations: externally for gross abnormalities, pup body weight, sex ratio, clinical signs

Statistics:

Dunnett, Fisher`s exact, Dunn, Mann-Whitney, Wilcoxon tests.

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Lower bw gain at 1000 mg/kg bw in females during gestation and lactation; no effect in males and in females before pregnancy. The lower bw gain did not affect reproductive outcome and offspring and was therefore considered as not adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Lower bw gain at 1000 mg/kg bw in females during gestation and lactation; no effect in males and in females before pregnancy. The lower bw gain did not affect reproductive outcome and offspring and was therefore considered as not adverse.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Accumulation of the sex-linked alpha-2-µ-globulin was noted in 300 and 1000 mg/kg bw/day treated males. This effect is not of toxicological relevance for humans.

Other effects:

no effects observed

Description (incidence and severity):

Test substance intake: At 1000 mg/kg bw/day slightly lower food consumption in females during gestation, no effect in males and females before pregnancy. The lower food consupmtion did not affect reproductive outcome and offspring and was therefore considered as not adverse.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The reduced mating index was not correlated to pathologic changes in the reproductive organs and thus considered accidental.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No deaths occurred in any group, which were attributed to treatment with the test substance. In the male and female treated groups, there were no clinical signs, which were considered to represent adverse effect of the test substance in any animal.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Males: The food consumption of all the treated males was similar to that of the control males, during the pre-mating period. The body weight change of the treated males was similar to that of the control animals.
- Females: The food consumption of all the treated females was similar to that of the control females, during the pre-mating and post-mating periods except for females of the 1000 mg/kg/day group in which a slight reduction in food consumption was recorded during the pregnancy (day 0 to day 20 of pregnancy: -6%, not statistically significant). This difference correlating with an effect on body weight gain was considered to be related to treatment with the test substance. The body weight change of the females given 100 or 300 mg/kg bw/day was similar to that of the control group during the pre-mating , pregnancy and lactation periods. In the 1000 mg/kg/day treated group, the body weight change was similar during the pre-mating period , but was slightly lower during pregnancy (day 0 to day 20 of pregnancy: - 10%, not statistically significant) and lactation (day 1 to day 4 post-partum: -38%, not statistically significant). Mean body weights on day 0 of pregnancy and day 4 post-partum statistically differed from that of the control group. These differences, correlating with a reduction of food consumption, (during the pregnancy period) was considered to be not adverse, since the effect was observed in pregnant females solely and did not affect the reproductive outcome and the offspring.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
The pre-coital interval was similar in the control and the treated groups. All paired animals (except one control pair) mated within 1 to 4 days of cohabitation, i .e. within the duration of a single estrous cycle. No particular microscopic finding was noted in the treated ovaries as their morphological characteristics were normal (particularly the number of ovarian follicles and corpora lutea). Hyalinosis of the blood vessels, haemosiderosis and sometimes capillary haemorrhage, were seen with similar incidence and/or severity in the uterus of the treated and control females.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No treament-related abnormalities were found in the treated animals. The tailed and round spermatids were unaffected and the different stages of spermatogenic cycle were undisturbed by treatment with the test substance. In almost all treated as well as control males, a few degenerated necrotic cells sloughed in the lumen (minimal or slight), multinucleated giant cells (minimal), vacuoles of the seminiferous tubules (minimal or slight) were noted with similar incidence and/or severity in all control and treated groups. These findings with such severity are commonly recorded as spontaneous changes in the rat and were considered to be of no toxicological importance. The seminiferous tubules were lined with Sertoli cells only (minimal or slight) in 1/10 males given 300 mg/kg bw/day and in 2/10 males given 1000 mg/kg bw/day. For a third male from the same group, tubules lined with Sertoli cells only were considered to be tubuli recti as they were situated beneath the capsule. For 1 /10 males given 300 mg/kg bw/day and anothergiven 1000 mg/kg bw/day, minimal reduction in the number of spermatids was observed in very few seminiferous tubules. Minimal vacuolization of Sertolicells was observed in 1/10 males given 1000 mg /kg bw/day. Although not found in the control males, these microscopic abnormalities recorded with minimal severity in few or very few seminiferous tubules of a few males were considered to be without relationship to the treatment and most probably fortuitous.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The mating index was 100% for the pairs of the control, 100 and 300 mg/kg bw/day groups. The mating index was lower in the 1000 mg /kg bw/day group: 7/10 pairs (70%) mated within the two weeks of cohabitation. As no treatment-related morphological changes were noted in the genital organs of the high-dose male and female animals, this finding is considered to be accidental. The fertility index (pregnant/mated) was similar in the control and the treated groups, ranging from 90 to 100% without indication of dose- or treatment-relationship.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No compound-related differences in organ weights were noted.

GROSS PATHOLOGY (PARENTAL ANIMALS)
The few macroscopic observations recorded were those commonly encountered in the laboratory rat of this strain and age and thus considered to be of no toxicological importance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There appears to be a dose-related higher severity of acidophilic globules in the cortical tubular epithelium of the kidneys of the treated male rats, given 300 and 1000 mg/kg bw/day. As no tubular degeneration/necrosis was observed in the kidneys of the males of any group, the presence of acidophilic globules was considered to be of minor toxicological importance and most probably due to the accumulation of the sex-linked alpha 2 micro-globulin in the cortical tubular cells of the male rats. This is a sex- and species-specific effect, as no such accumulation occurs in female rats or in humans. Thus, this finding has no relevance for humans in terms of risk assessment.

Dose descriptor:

NOEL

Remarks:

systemic

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: histopathology: higher severity of acidophilic globules in the kindneys due to accumulation of alpha-2-u-globulin. As a sex-and species-specific effect of male rats, this finding has no relevance for humans

Dose descriptor:

NOEL

Remarks:

systemic

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: reduced food consumption and body weight gain in dams during gestation and lactation

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: taking into account that alpha-2-u-globulin accumulation in male rats is not relevant for humans and body weight gain reduction in females did not affect the reproductive outcome

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
The number of pups which died during the four days of observation after birth was low (0 to 5 %) and similar in all the groups.

CLINICAL SIGNS (OFFSPRING)
There were no notable clinica l signs in the pups of the control or treated groups after birth.

BODY WEIGHT (OFFSPRING)
The weights of pups were similar in the control and the treated groups on day 1 and day 4 post-partum (being slightly higher in the high -dose group, as expected for lower-sized litters) .

GROSS PATHOLOGY (OFFSPRING)
There were no gross external abnormalities in the control and the treated groups.

Reproductive effects observed:

not specified

Conclusions:

The test item had no effect on reproductive performance.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 2) and consistent studies from a reference substance with similar structure and intrinsic properties. Read-across is justified based on common functional groups, common breakdown products, and similarities in PC/ECOTOX/TOX properties (refer to endpoint discussion for further details).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Justification for read-across

No sufficient data are available for the toxicity to reproduction of 6-methyl-2-oxoperhydropyrimidin-4-ylurea (Crotodur, CAS 1129-42-6). In order to fulfil the standard information requirements set out in Annex VIII, 8.5, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from a structurally related substance was conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

Toxicity to Reproduction

CAS	1129-42-6 TARGET SUBSTANCE	6104-30-9
Chemical Name	6-methyl-2-oxoperhydropyrimidin-4-ylurea (Crotodur)	N,N”-(2-methylpropane-1,1-diyl) diurea (Isodur)
MW	172.1851 g/mol	174.2010 g/mol
Toxicity to Reproduction - oral	RA: CAS 6104-30-9	NOAEL ≥ 1000 mg/kg bw/day

 

The above mentioned substances are considered to be similar on the basis of structural similarity resulting in similar properties and/or activities. The available endpoint information is used to predict the same endpoints for 6-methyl-2-oxoperhydropyrimidin-4-ylurea (Crotodur, CAS 1129-42-6).

A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

 

Discussion

 

Toxicity to Reproduction - oral

No data are available for 6-methyl-2-oxoperhydropyrimidin-4-ylurea (Crotodur, CAS 1129-42-6), however, 2 reliable studies are available for the structurally analogue substance N,N”-(2-methylpropane-1,1-diyl) diurea (Isodur, CAS 6104-30-9), which were used for read-across based on the analogue approach.

In the available key study (CIT, 2003b) the structural analogue substance CAS 6104-30-9 was investigated for toxicity to reproduction in a one-generation study. The study was conducted similar to the OECD test guidelines 416 and 421, and in compliance with GLP. 25 Sprague-Dawley rats per sex per dose received the test substance in carboxymethyl cellulose via gavage at doses of 100, 300, and 1000 mg/kg bw/day. The dose-levels were selected on the basis of available toxicological data obtained in a combined repeated dose toxicity/developmental toxicity screening test (CIT, 2003a) with the test item: 600 mg/kg bw/day as the expected NOAEL, 1200 mg/kg bw/day as the maximum feasible dose-level. Males were treated throughout the pre-mating period (10 weeks), mating period (2 weeks), and until sacrifice; the females received the test material throughout the pre-mating period (10 weeks), mating period (2 weeks), and pregnancy until day 14 post-coitum inclusive. Treatment was carried out daily on 7 days per week, and concurrent controls received the vehicle only. For mating the animals were cohoused in ratios of 1 male/1 female per dose group during the night, until mating occurred or 14 days elapsed. Pregnancy was proved by vaginal plug or sperm in vaginal smear and referred to as Day 0 of pregnancy. Cage side observations were conducted at least twice a day, and detailed clinical observations were performed at least once daily. Body weights of each male were recorded once a week until sacrifice, and of each female once a week during the pre-mating and mating periods, then on days 0, 7 and 15 post-coitum. Males were examined for testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, sperm motility, and sperm morphology, while in females for technical and scientific reasons, the estrous cycle was not taken into account for the interpretation of the data. Reproductive indices calculated were mating data, male fertility data, and female fertility data (female fertility and gestation indices, hysterectomy parameters). At study termination, the animals were sacrificed. The time schedule was for females, which did not mate, at least one week after the end of the mating period, for all surviving maternal animals on Day 15 post coitum, and for all surviving males after most hysterectomies of the females had been performed. Gross necropsy consisted of a macroscopic post-mortem examination of the principal thoracic and abdominal organs (with particular attention paid to the reproductive organs) and was performed on all animals, including those that died during the study or were killed prematurely. For all females, the number of corpora lutea and implantation sites was recorded whenever possible. No microscopic examination was deemed necessary since all the macroscopic lesions were considered to be unrelated to the treatment. The body weight of all animals killed at terminal sacrifice was recorded and the following organs were weighed (wet) as soon as possible after dissection. For all males these organs were testes, epididymides, prostate, seminal vesicles together with coagulating glands, pituitary gland, and adrenals; testes and epididymides were weighed separately. In all females uterus, ovaries, pituitary gland, and adrenals were weighed. The pups were discarded without further investigation. The study did not reveal any treatment-related effects on neither systemic toxicity nor reproductive function. In females, effects on food consumption and body weight gain were noted: during pregnancy (days 1 to 15), minimal (-7%) and slight (-14%, p<0.01) decreases in food consumption were recorded at the 600 and 1200 mg/kg/day dose-levels, respectively. However, during pre-mating (days 1 to 71), the food consumption was similar in the control and the treated females. The body weight gain at 600 mg/kg/day was similar to that of the controls during the pre-mating period (days 1 to 71), while it was slightly lower (-11 %, p<0 05) in females given 1200 mg/kg/day. During pregnancy (days 1 to 15), a similar moderate decrease in body weight gain was recorded in females given 600 (-24%, p<0.001) and 1200 mg/kg/day (-24%, p<0.001).

These data are further supported by a screening study, conducted according to OECD 422 and in compliance with GLP, with the structural analogue substance CAS 6104-30-9 (CIT, 2003a). 10 Sprague-Dawley rats per sex per dose received the test item in carboxymethyl cellulose via gavage at doses of 100, 300, and 1000 mg/kg bw/day. Males were treated throughout pre-mating (15 days), and during the mating and post-mating periods until sacrifice (34 days in total). Females received the test material throughout pre-mating (15 days) and mating period, during pregnancy and lactation, and until day 4 post partum. Treatment was conducted once daily for 7 days per week; concurrent controls received the vehicle only. In the high dose group (1000 mg/kg bw/day) slightly lower food consumption was noted during gestation (-6%, compared to controls, day 0-day 20 of pregnancy), in combination with slightly lower body weight gain during gestation (-10%, compared to controls, day 0-day 20 of pregnancy) and lactation (-38%, compared to controls, day 1-day 4 post-partum). At necropsy, no test material related changes were reported for the reproductive organs. The mating index calculated was 100% for the pairs of the control, 100 and 300 mg/kg bw/day groups. The mating index was lower in the 1000 mg /kg bw/day group: 7/10 pairs (70%) mated within the two weeks of cohabitation. As no treatment-related morphological changes were noted in the genital organs of the high-dose male and female animals, this finding is considered to be accidental. The fertility index (pregnant/mated) was similar in the control and the treated groups, ranging from 90 to 100% without indication of dose- or treatment-relationship. External examinations of the litters (all animals per litter) revealed no indications for a substance-induced developmental toxicity up to and including the highest tested dose of 1000 mg/kg bw/day.

In summary, both studies revealed solely effects on food consumption and body weight gain in females. However, both studies revealed further that only pregnant and lactating females were affected; no such observation was made for the females during the pre-mating period or for the males. The reduced body weight gain is considered to be a secondary effect to the decreased food consumption. Based on the fact that only pregnant females of the dose groups treated with 600 mg/kg bw/day and above showed reduced food consumption, this is expected to be the result of commonly observed abnormal taste and smell perception during pregnancy, which was reported not only for rodents (Clarke, S. N. and Bernstein, I. L., 2001), but is also widely known for humans (Klimack-Nawrot, E., et al., 2012; Nordin, S., et al., 2004; Poothullil, J.M., 1995). It can therefore be expected that reduced food consumption is a consequence of abnormal taste and smell during gestation and may be a foeto-protective mechanism to avoid poisoning (Nordin, S., et al., 2004). This protective mechanism is expected to be enhanced by treatment with the test item, as a dose relationship was observed. Since this did affect neither the reproductive performance, nor the viability and development of the offspring, this effect was considered to be of no toxicological relevance.

In conclusion, there were no substance-related effects on the male and female reproductive performance. The lower mating index noted in the screening study (CIT, 2003a) could not be confirmed in the one-generation study (CIT, 2003b) and is therefore considered to be incidental and not related to the test substance. In the light of the results of both studies available, the NOAEL for systemic toxicity was set at ≥ 200 mg/kg bw /day for both males and females. Based on the fact that only in the screening study (CIT, 2003a) the offspring was examined and the no-effect of the decreased body weights on the pup development was proven, the NOAEL for reproduction was set at ≥1000 mg/kg bw/day.

 

References:

Clarke, S. N. and Berrnstein, I. L. (2001). NaCL increases during pregnancy and lactation: assessment using brief access tests. Pharmacol Biochem Behav. 68(3): 555-563

Klimacka-Nawrot, E., Suchecka, W., Hartmann, M., Gałazka, A., Musialik, J., Petelenz, M. (2012). Changes id food preferences in pregnant women. Wiad Lek. 65(1): 10-14

Nordin, S., Broman, D. A., Olofsson, J. K., Wulff, M. (2004). A longitudinal Descriptive Study of Self-reported Abnormal Smell and Taste Perception in Pregnant Women. Chem. Senses 29: 391-402

Poothullil, J. M. (1995). Neurosci Biobehav Rev. 19(3): 407-412

 

 

Toxicity to Reproduction - inhalation

No data available.

 

Toxicity to Reproduction - dermal

No data available.

Short description of key information:
Oral ( OECD 422, RA from CAS 6104-30-9), rat: NOEL (systemic, female)=300 mg/kg bw/day, NOAEL (systemic, male, female)>=1000 mg/kg bw/day, NOAEL (reproduction, male/female)>=1000 mg/kg bw/day
Oral
Inhalation: No data available.
Dermal: No data available.

Justification for selection of Effect on fertility via oral route:
Hazard assessment is conducted by means of read-across from a structural analogue. The selected study is the most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall quality assessment (refer to the endpoint discussion for further details).

Justification for selection of Effect on fertility via inhalation route:
Reliable data are available for the oral route.

Justification for selection of Effect on fertility via dermal route:
Reliable data are available for the oral route.

Effects on developmental toxicity

Description of key information

Oral (OECD 414, RA from CAS 6104-30-9), rat: NOAEL (maternal)=1000 mg/kg bw/day, NOAEL (developmenta toxicity/teratogenicity)=1000 m/kg bw/day
Inhalation: No data available.
Dermal: No data available.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1991-07-17 to 1991-08-09

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted similar or equivalent to the appropriate OECD test guideline with acceptable restrictions. The restriction was that the test animals were treated with the test item up to day 16 post coitum only. The study was compliant with GLP. Since this is a read-across from a structural analogue substance (CAS 6104-30-9), the reliability was set from RL1 to RL2.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(2001)

Deviations:

yes

Remarks:

(the animals were treated with the test item only up to day 16 post coitum)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Wistar rats (Chbb:THOM (SPF))
- Source: Karl Thomae, Biberbach an der Riss, Germany
- Age at study initiation: 65-74 days (sexually mature, virgin)
- Weight at study initiation: mean bw approximately 225.4 g
- Fasting period before study: none
- Housing: individually in type DK III stainless steel wire mesh cages, floor area approximately 800 cm² (supplied by Becker & Co, Castrop-Rauxel, Germany)
- Diet: ground Kliba 343 feed rat/mouse/hamster (Klingentalmuehle AG, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was freshly prepared as a suspension in 0.5% aqueous CMC solution shortly before administration.

VEHICLE
- 0.5% CMC aqueous CMC solution (Tylose CB 30.000; purified carboxymethyl cellulose supplied by Hoechst AG, Frankfurt/Main, Germany)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance suspensions up to 3 h were carried out during the study. Furthermore, samples of the test substance suspensions were sent to the analytical laboratory of the BASF AG twice during the study period for verification of concentrations. The samples which were sent for the first concentration control analyses toward the beginning of the administration period were also used to verify the homogeneity for the samples of the low and high concentrations (100 mg/kg bw and 1000 mg/kg bw). 6 samples (2 from the top, middle, and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running.
The test substance analysis was carried out by using HPLC.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1-4 untreated females with 1 untreated fertile male
- Length of cohabitation: from 16.00 h afternoon to 7.30 h the next mroning
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

on gestation days 6-15

Frequency of treatment:

once daily

Duration of test:

On day 20 post coitum all animals were sacrificed.

Remarks:

Doses / Concentrations:
100, 400, and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the outcome of a preliminary range-finding study

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: observations for mortality was made twice a day on working days and once a day on Saturdays, Sundays and public holidays; observations for clinical signs were made at least once daily on days 0-20 post coitum; more often when clinical sysmptoms of toxicity were elicited

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: on days 0, 1, 3, 6, 8, 10, 13, 15, 17, and 20 post coitum

FOOD CONSUMPTION: Yes

WATER CONSUMPTION: No
- Time schedule for examinations:

HAEMATOLOGY: Yes
- Time schedule for examinations: on day 16 post coitum
- Parameters checked: white blood cell count (total and differential), red blood cell count, haemoglobine, haematocrite, mean corpuscular volume, mean corpuscular haemoglobine concentration, platelet count, thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for examinations: on day 16 post coitum
- Parameters checked: alanine aminotranfrease (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), serum-gamma-glutamyltransferase (SGGT), electrolytes (Na, K, Cl, inorg. Phosphate, Ca, Mg), urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol

URINALYSIS: Yes
- Time schedule for examinations: on day 16 post coitum
- Parameters checked: nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, volume, secific gravity, sediment (fat, crystals, cells: renal tubular, transitional, sqamous, casts, erythrocytes, leucocytes, bacteria)

POST-MORTEM EXAMINATIONS: Yes (gross pathology)
- Sacrifice on gestation day 20

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
- Number of dead foetuses (hypoxemic foetuses which did not breath spontaneously after the uterus had beenopened): Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: No

Statistics:

Dunett's Test was used for statistical evaluation of food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, weight of foetuses, weight of placentae, corpura lutea, implantations, pre- and postimplantation losses, resorptions, and live foetuses.
Fisher's Exact Test was used for statistical evaluation of conception rate, mortality of the dams, and all foetal findings.

Indices:

- conception rate (%) calculted by (number of pregnant animals / number of fertilised animals) x 100
- preimplantation loss (%) calculated by ((number of corpura lutea - number of implantations) / number of corpura lutea) x 100*
- postimplantation loss (%) calculated by ((number of implantations - number of live foetuses) / number of implantations) x 100

* calculation on the basis of each individual pregnant animal with scheduled sacrifice

Historical control data:

Historical control data were available.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
FERTILITY OF THE DAMS
The following number of females were not pregnant after the mating procedure:
- vehicle control group: 2/25
- 100 mg/kg bw/day group: 3/25
- 400 mg/kg bw/day group: 3/25
- 1000 mg/kg bw/day group: 1/25

CLINICAL SIGNS AND MORTALITY OF THE DAMS
- There were no mortalities in any of the groups throughout the study period.
- There were no abnormal clinical findings in any dam of any group throughout the study period.

FOOD CONSUMPTION OF THE DAMS
- The food consumption of all test groups (100, 400, and 1000 mg/kg bw/day) did not show any differences of biological relevance as compared to the control group. All values were within the range of biological variation.

BODY WEIGHT AND BODY WEIGHT GAIN OF THE DAMS
- the body weights and body weight gains of the dams of the test groups were similar to those of the control group. All differences observed between the groups were without any biological relevance.
- The result of the corrected body weight gain (terminal body weight on day 20 post coitum minus weight of the uterus before it was opened minus body weight on day 6 post coitum) of all substance treated groups did not show any differences of biological relevance if compared to the controls.

HAEMATOLOGY
No statistically significant changes were reported.

CLINICAL CHEMISTRY
No statistically significant changes were reported.

URINALYSIS
No statistically significant changes were reported.

NECROPSY OF THE DAMS
- The uterus weights of the animals were not influenced by test material administration. The differences between the groups were without biological relevance.

REPRODUCTION DATA OF THE DAMS
- The conception rate varied between 88% (100 and 400 mg/kg bw/day) and 96% (1000 mg/kg bw/day). There were no substance-related and/or statistically significant differences of biological relevance between the groups in conception rate, in the mean number of corpura lutea and implantation sites, in the values calculated for the pre- and postimplantation losses, or in the number of resorptions and viable foetuses. The differences evident were considered incidental and within the normal range of deviations for animals of this strain and age.

WEIGHT OF PLACENTAE
- The mean placental weights in the test groups were comparable to the actual control values.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
SEX DISTRIBUTION OF THE FOETUSES
- The sex distribution of the foetuses in the test groups was comparable with the control foetuses. The differences observed in comparison to the control were without any biological relevance.

WEIGHT OF FOETUSES
- The mean foetal weight were not influenced by the test substance administration. All values were within the range of biological variation.

EXTERNAL EXAMINATIONOF THE FOETUSES
- External malformations were recorded for one low dose foetus only (100 mg/kg bw/day). For this foetus, brachygnathia and aglossostomia were noted. These or very similar malformations were also present in the historical control data of the breeder at a low frequency. Therefore, and because these external malformations (in just one low dose foetus) show no relationship to dosing, they were finally assessed as being of spontaneous origin. No further variations were observed in the foetuses in any group at the external examination.
- So-called unclassified observations (placentae fused or necrobiotic) were recorded for 2 control, 4 low dose (100 mg/kg bw/day), and 2 mid dose foetuses (400 mg/kg bw/day), but not for any high dose foetus (1000 mg/kg bw/day).

SOFT TISSUE EXAMINATION OF FOETUSES
- The examination of the organs of the foetuses revealed one foetus with malformations in the low and mid dose each: hydrocephaly together with microphthalmia (left) and anophthalmia (right) occurred in one foetus of the low dose (100 mg/kg bw/day) and dextrocardia was recorded for one mid dose foetus (400 mg/kg bw/day). Due to the missing dose-response relationship and due to the fact that these malformations were also present in the historical control data, they were regarded as being of sponatneous nature and not related to test material administration.
- Variations (dilated renal pelvis and/or hydrourether) were detected in all groups without any statistically significant differences between the groups. All values were fully within the range of biological variation.
- No so-called unclassified observations (like blood coagulum araound the bladder) were recorded.

SKELETAL EXAMINATION OF THE FOETUSES
- Various skeletal malformations were seen in a number of foetuses of alll groups. These malformations were related to the skull (mandible fused or various skull abnormalities), the vertebral column (thoracic/lumbar vertebral body/bodies dumbell-shaped (asym.) or bipartite (asym.)), the sternum (sternebra(e) bipartite, ossification centers dislocated) and/or the ribs (fused ribs). Most of the described skeletal malformations occurred without any relation to dosing and without any statistically significant differences between the groups; however, the foetal and/or litter incidences for two malformations of the skeleton (thoracic/lumbar vertebral body/bodies dumbell-shaped (asym.); sternebra(e) bipartite, ossification centers dislocated) and the overall number of skeletal malformations were increased in the substance treated groups, but without a clear dose-response relationship. All values, however, are fully within or just above the historical control range. It is notable that the control values were unexpectedly low and that the relevant values for the substance-treated groups do show a clear dose-response relationship. Therefore, the statistically significanly, but not dose-relatedly, increased foetal and/or litter incidences concerning skeletal malformations were finally considered random.
- The variations elicitated were related to the ribs (shortened 13th, accessory 14th ribs or rudimentary cervical ribs), the vertebral column (accessory thoracic vertebra) and the sternum (sternebra(e) of irregular shape or bipartite) and were found in all groups without any dose-response relationship. The statitically significant increase of sternebra(e) of irregular shape (100 mg/kg bw/day) and the low occurrence of rudimentary cervical rib(s) (400 mg/kg bw/day) compared to controls were without any biological relevance; the diffrernces occurred without any relation to dosing and the values were fully within the range of the historical control.
- In all groups signs of retardations (incomplete or missing ossifications of vertebral bodies/arches, sternebra(e), skull and/or the hyoid bone) were found without any differences of biological relevance between the groups. The statistically significantly lower number of low dose foetuses (100 mg/kg bw/day) with sternebra(e) with only one ossification center and of high dose foetuses (1000 mg/kg bw/day) with not ossified sternebra(e) were of spontaneous nature; the relevant values were fully within the range ofthe historical control.


SUMMARY
In summary, there were no statistically significant differences between the control and the test groups concerning foetal external and soft tissue examinations. Only the evaluation of the foetal skeletons revealed statistically significant increase in the malformation rate in the low and mid dose groups (100 and 400 mg/kg bw/day), but not in the high dose group (1000 mg/kg bw/day). The statistically significant differences between the control and the low and mid dose groups were not test material related, but were of spontaneous nature due to unexpected low occurrence of dumbell-shaped (asym.) thoracic vertebral body/bodies and/or bipartite sternebra(e) with dislocated ossification centers in the control group. Moreover, the slightly, but statistically significantly increased number of low dose foetuses with skeletal variations was assessed as being of spontaneous nature due to missing dose-response relationship.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Tab. 1: Selected foetal skeletal malformations

			control	100 mg/kg bw/day	400 mg/kg bw/day	1000 mg/kg bw/day
Thoracic vertebral body/bodies dumbbell-shaped (asym.)	Foetal incidence	N	0	5*	10**	5*
		%	0.0	3.2	6.6	3.0
	Litter incidence	N	0	4*	7**	4
		%	0.0	18.0	32.0	17.0
Sternebra(e) bipartite, ossification center dislocated	Foetal incidence	N	0	1	4*	2
		%	0.0	0.6	2.6	1.2
	Litter incidence	N	0	1	3	2.
		%	0.0	4.5	14.0	8.3

statistical significance indicated as * p<0.05; ** p<0.01

Tab. 2: Incidence of selected skeletal malformations observed in the study compared to the historical control data

	Actual foetal incidence (%)		Historical foetal incidence (%)	Actual litter incidence (%)		Historical litter incidence (%)
Thoracic vertebral body/bodies dumbbell-shaped (asym.)	control 100 mg/kg bw/day 400 mg/kg bw/day 1000 mg/kg bw/day	0.0 3.2 6.6 3.0	0-8.8	control 100 mg/kg bw/day 400 mg/kg bw/day 1000 mg/kg bw/day	0.0 18.0 32.0 17.0	0-39.1
Sternebra(e) bipartite, ossification center dislocated	control 100 mg/kg bw/day 400 mg/kg bw/day 1000 mg/kg bw/day	0.0 0.6 2.6 1.2	0-2.2	control 100 mg/kg bw/day 400 mg/kg bw/day 1000 mg/kg bw/day	0.0 4.5 14.0 8.3	0-14.3

Conclusions:

The test item had no effect on intrauterine development.

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:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 2) and consistent studies from a reference substance with similar structure and intrinsic properties. Read-across is justified based on common functional groups, common breakdown products, and similarities in PC/ECOTOX/TOX properties (refer to endpoint discussion for further details).

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

Justification for read-across

No sufficient data are available for developmental toxicity/teratogenicity of 6-methyl-2-oxoperhydropyrimidin-4-ylurea (Crotodur, CAS 1129-42-6). In order to fulfil the standard information requirements set out in Annex VIII, 8.5, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from a structurally related substance was conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

Developmental Toxicity/Teratogenicity

CAS	1129-42-6 TARGET SUBSTANCE	6104-30-9
Chemical Name	6-methyl-2-oxoperhydropyrimidin-4-ylurea (Crotodur)	N,N”-(2-methylpropane-1,1-diyl) diurea (Isodur)
MW	172.1851 g/mol	174.2010 g/mol
Developmental Toxicity/Teratogenicity - oral	RA: CAS 6104-30-9	NOAEL ≥ 1000 mg/kg bw/day

 

The above mentioned substances are considered to be similar on the basis of structural similarity resulting in similar properties and/or activities. The available endpoint information is used to predict the same endpoints for 6-methyl-2-oxoperhydropyrimidin-4-ylurea (Crotodur, CAS 1129-42-6).

A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

 

Discussion

 

Developmental Toxicity/Teratogenicity - oral

No data are available for 6-methyl-2-oxoperhydropyrimidin-4-ylurea (Crotodur, CAS 1129-42-6), however, a reliable study is available for the structurally analogue substance N,N”-(2-methylpropane-1,1-diyl) diurea (Isodur, CAS 6104-30-9), which was used for read-across based on the analogue approach.

 

In the available key study (BASF AG, 1993) the structural analogue substance CAS 6104-30-9 was investigated for developmental toxicity/teratogenicity. The study was conducted according to OECD 414 and in compliance with GLP. 25 naive female Wistar rats were mated in a ratio of 1/4 with respective naive males by cohousing overnight, and treated with the test substance via gavage from gestation days 6-15. The test material was administered as a suspension in 0.5% aqueous carboxymethyl cellulose solution at doses of 100, 400, and 1000 mg/kg bw/day once daily on 7 days/week. The doses applied were selected on the basis of the outcome of a preliminary range-finding study. There were no substantial, substance-related effects on the dams concerning food consumption, body weights, body weight changes, uterine weights, corrected body weight changes, clinical and necropsy observations up to and including a dose of 1000 mg/kg bw/day. There were no differences of of biological relevance between the control and the test groups in conception rate, mean number of corpura lutea, total implantations, resorptions and live foetuses, foetal sex ratio, or in the values calculated for the pre- and the post-implantation losses. No dose- and/or substance-related differences were recorded for placental and foetal body weights. The external, soft tissue, and skeletal examinations of the foetuses revealed no differences between the control and the test groups which might be related to test material administration. Number and type of foetal external, soft tissue, and skeletal findings, which were classified as malformations, variations and/or retardations, recorded for the low, mid and high dose foetuses were substantially similar to actual and/or historical control values. Thus, under the conditions of this study, the test item CAS 6104-30-9 caused no signs of maternal toxicity and no signs of embryo-/fetotoxicity up to and including a dose of 1000 mg/kg bw/day. There were no indications of teratogenic effects, which could be causally related to the test substance administration. Thus, the NOAEL for both the maternal and foetal organism was set at ≥ 1000 mg/kg bw.

 

Developmental Toxicity/Teratogenicity - inhalation

No data available.

 

Developmental Toxicity/Teratogenicity - dermal

No data available.

Justification for selection of Effect on developmental toxicity: via oral route:
Hazard assessment is conducted by means of read-across from a structural analogue. The selected study is the most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall quality assessment (refer to the endpoint discussion for further details).

Justification for selection of Effect on developmental toxicity: via inhalation route:
Reliable data are available for the oral route.

Justification for selection of Effect on developmental toxicity: via dermal route:
Reliable data are available for the oral route.

Toxicity to reproduction: other studies

Additional information

No data available.

Justification for classification or non-classification

The available data are reliable and suitable for classification. Based on these data, classification for toxicity to reproduction according to Regulation (EC) 1272/2008 or Directive 67/548/EEC is not warranted.