N,N''-methylenebis[N'-[3-(hydroxymethyl)-2,5-dioxoimidazolidin-4-yl]urea]

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Short description of key information:

In accordance with section 1 of REACH Annex XI, testing for reproductive toxicity in animals is not scientifically necessary and is therefore not required.

Several subchronic and subacute toxicity tests performed with the test substance have clearly demonstrated low systemic toxicity. In particular, no effects on weight or microstructure of rat testes or ovaries were found when rats were dosed orally for 90 days at dosages up to 200 and 300 mg/kg/day. The observed pattern of toxicity shows a direct contact effect in the stomach, but little indication of systemic toxicity. In one sub-chronic study (performed under GLP and following a standard test method) micropathology investigation of rats dosed at 300 mg/kg/day found: "The degree of spermatogenesis in the testes of the high dose males was similar to their counterpart vehicle controls.  The ovarian activity of the high dose and vehicle controls was similar but varied depending on the stage of the estrus cycle. Dilatation of the uterine lumen and uterine glands occurred in individual rats in the vehicle control and high dose females and reflected the stages of the estrus cycle." Futher, a clear absence of developmental toxicity has been demonstrated in rat studies using two different routes of administration.

These results show that there is no reason to require further investigation of reproductive performance in a 1- or 2-generation study, or performance of a reprotoxicity screening study: indeed low toxicity to reproduction can reasonably be predicted.  Hence, there is sufficient weight of evidence, to claim that additional animal testing for assessment of effects on fertility is not justified according to article 13.1 of the REACH regulation, as well as not justified regarding reduction of testing on vertebrate animals in the spirit of Directive 2010/63/EU. In addition, the eventual cosmetic use of the substance argues against the performance of an extensive animal study (Directive 76/768/EEC).

Justification for selection of Effect on fertility via oral route:

Study not scientifically necessary and therefore waived in accordance with article 13.1 of the REACH regulation and REACH Annex XI, Section 1 (justification supplied).

Effects on developmental toxicity

Description of key information

No evidence of teratogenic  or embryo-/foetotoxic activity has been observed in a well conducted test of developmental toxicity performed using a close chemical analogue of the registered substance

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Adequately reported study performed using a standard test method: includes sufficient information to permit evaluation of developmental toxicity.

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

GLP compliance:

no

Remarks:

Pre-GLP study

Limit test:

no

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

Virgin adult females

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Daily oral administration during the dosing period.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Mated with young adult male mice: observation of vaginal sperm plug confirmed mating and was designated Day 0 of gestation.

Duration of treatment / exposure:

10 days (gestation Days 6-15)

Frequency of treatment:

Once daily

Duration of test:

Females terminated on gestation Day 17.

Remarks:

Doses / Concentrations:
30, 95, 300 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

At least 25 females were mated and dosed in each group (25-27: pregnant and surviving to termination 20-22/group).

Control animals:

yes, sham-exposed

other: positive controls, given aspirin at 150 mg/kg

Maternal examinations:

On gestation Day 17 dams terminated and subjected to caesarean section. Urogenital tracts examined for abnormality.

Ovaries and uterine content:

Implantation and resorption sites counted, plus live and dead foetuses.

Fetal examinations:

Live foetuses weighed. All foetuses sexed and examined for external abnormality, then examined in detail:
- one third of each litter examined for visceral/soft tissue abnormalities (x10 magnification used to aid inspection)
- remainder processed for skeletal examination (KOH then alizarin red stain).

Statistics:

Results tabulated/dam:
- implant sites
- resorption sites
- Live and dead foetuses
- male/female embryos
- average foetus weight/litter.

Indices calculated per group:
- live litters
- implant sites: total and average/dam
- resorptions: total number, dams with 1 or more, dams with complete resorption, % with 1 or more, % with complete resorption
- live foetuses: total and average/dam, sex ratio
- dead foetuses: total number, dams with 1 or more, dams with all dead, % with 1 or more, % with all dead
- average foetus weight.

Skeletal abnormality/variant indices:
-

Indices:

Results tabulated/dam:
- implant sites
- resorption sites
- Live and dead foetusess
- male/female embryos
- average foetus weight/litter.

Indices calculated per group:
- live litters
- implant sites: total and average/dam
- resorptions: total number, dams with 1 or more, dams with complete resorption, % with 1 or more, % with complete resorption
- live foetuses: total and average/dam, sex ratio
- dead foetuses: total number, dams with 1 or more, dams with all dead, % with 1 or more, % with all dead
- average foetus weight.

Skeletal abnormality/variant categories (reported/group as affected foetus number and number of litters with one or more affected)
- sternebrae (incompete ossification, bipartite, extra or missing)
- ribs (fused/split, wavy, >13)
- vertebrae (incomplete ossification)
- skull (incomplete closure)
- extremities (incomplete ossification, missing)
- hyoid (missing, reduced).

Soft tissue abnormalities: none observed, so not categorised.

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:

not specified

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects. Remark: No adverse reactions to treatment are reported. Bodyweight data show no effect of treatment.

Details on maternal toxic effects:
Bodyweight increase over the test period was closely similar in test groups and the sham treatment controls (51-62% increase over 17 days)

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

clinical signs

gross pathology

mortality

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

not specified

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No pattern of increased skeletal abnormality or variation was seen in test groups when compared to concurrent controls. A 28% incidence of >+13 ribs in live foetuses of the 30 mg/kg/day test group compared to 12% in controls was not considered biologically significant in the absence of a similar increase at higher test dosages.

Visceral malformations:

not specified

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No pattern of increased skeletal abnormality or variation was seen in test groups when compared to concurrent controls. A 28% incidence of >+13 ribs in live foetuses of the 30 mg/kg/day test group compared to 12% in controls was not considered biologically significant in the absence of a similar increase at higher test dosage.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: teratogenicity

Abnormalities:

no effects observed

Description (incidence and severity):

No pattern of increased skeletal abnormality or variation was seen in test groups when compared to concurrent controls. A 28% incidence of >+13 ribs in live foetuses of the 30 mg/kg/day test group compared to 12% in controls was not considered biologically significant in the absence of a similar increase at higher test dosage.

Developmental effects observed:

no

Maternal data

Group	Live litters	Mean implants/dam	Dams with ≥1 resorption site	Total resorptions	Mean live foetuses/dam	Dams with ≥1 dead foetus (% of dams)	Mean foetal weight (g)
Sham treated control	20	12.7	7	13	12.0	1 (5%)	0.89
Test: 30 mg/kg/day	20	11.6	12	23	10.3	3 (14.3%)	0.89
Test: 95 mg/kg/day	20	12.8	9	16	11.9	2 (10.0%)	0.87
Test: 300 mg/kg/day	20	12.1	6	25	10.8	3 (13.6%)	0.93
Aspirin positive control	21	12.0	15	29	10.4	6 (27.3%)	0.84

Soft tissue abnormalities: none observed.

Skeletal findings

Group	Live foetuses examined	Sternebrae	Ribs	Vertebrae	Skull	Extremities	Hyoid*
Sham treated control	167 (20)	55 N (19) 8 B (7) 18 M (4)	20 + (9)	7 N (3)	3 N (2)	8 N (2) 1 M (1)	23 R (10)
Test: 30 mg/kg/day	149 (20)	49 N (17) 5 B (4) 18 M (6)	42 + (12)	7 N (4)		8 N (4)	19 R (11)
Test: 95 mg/kg/day	165 (20)	75 N (19) 5 B (5) 31 M (10)	23 + (12)	6 N (2)	1 N (1)	4 N (2)	33 R (14)
Test: 300 mg/kg/day	165 (20)	65N (16) 7 B (5) 1 + (1) 14 M (6)	27 + (13)	5 N (3)		8 N (4)	17 R (11)
Aspirin positive control	158 (21)	97 N (19) 7 B (4) 33 M (11)	9 F (2) 1 W (1) 45 + (13)	16 N (6)	3 N (2)	16 N (8)	29 R (13)

(n) = number of litters concerned

N = incomplete ossification(skull: incomplete closure)   B = bipartite  M = missing   F = fused/split

+ = extra (sternebrae) or >13 (ribs)   W = wavy   R = reduced

* Hyoid also shown as “missing” in 22 control foetuses and up to 32, 52 and 37 test group foetuses (30, 95, 300 mg/kg/day respectively): subsequently stated to indicate lost samples, not available for inspection due to errors at necropsy

Conclusions:

Oral administration of test substance at up to 300 mg/kg/day produced no evidence of teratogenic effect and no clear evidence of embryotoxicity.

Reference 2

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Read-across from Klimisch 1 study performed on a close structural analogue which is manufactured in the same way (reaction of formaldehyde with a modified urea) and reacts with water in the same way (hydrolysis releasing formaldehyde) as the registered substance

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

80-120 days old at study start. Individually caged, in room designed to maintain 22+/-3C, 30-70% humidity, with a 12h light/dark cycle.

Route of administration:

dermal

Vehicle:

water

Remarks:

deionised

Details on exposure:

Applications sites shaved prior to first treatment and as necessary thereafter. Daily topical administration of a 30% solution onto mid-dorsal skin (4 x 4 cm site), without occlusion; rats collared during the treatment period to prevent ingestion.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Colourimetric analysis (samples heated with reagent under acidic conditions, absorption measured at 520 nm). Measured concentrations of two formulated dose samples (nominally 300 mg/ml) confirmed to be within 10% of nominal values, and test substance absence in vehicle controls confirmed.

Details on mating procedure:

Females mated 1F:1M or 2F:1M until vaginal plug or sperm was seen (Day 0).

Duration of treatment / exposure:

10 days (gestation days 6-15).

Frequency of treatment:

Daily

Duration of test:

Treated females terminated on day 20.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

27 females/group treated.

Control animals:

yes, concurrent vehicle

Maternal examinations:

Observed at least daily; bodyweights and food consumption measured during test period. Bodyweights and uterine weights recorded at necropsy.

Ovaries and uterine content:

Corpora lutea, early/late resorptions, viable/non-viable foetuses counted and recorded.

Fetal examinations:

Sexed, weighed, examined externally. One third of each litter stained for skeletal abnormalities, remainder prepared and examined for soft tissue abnormalities.

Statistics:

Analysis of variance (Dunnett's test); non-parametric test where variances differed significantly.

Indices:

Maternal bodyweights and food consumption.
Dams with resorptions only, dams with viable foetuses.
Viable foetuses/dam
Total implants/dam
Total implant losses/dam
Pre-implantation loss (corpora lutea - implants/corpora lutea), % by dam
Post-implantation loss (implants - viable foetuses/implants), % by dam.

Foetal sex distribution
Foetuses with soft tissue malformations
Foetuses with skeletal malformations
Foetuses with soft tissue and/or skeletal malformations.

Litters with with soft tissue malformations
Litters with skeletal malformations
Litters with soft tissue and/or skeletal malformations.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

Local skin reactions: yellowing, scabs and erythema (incidence dose-dependent).

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

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:

not examined

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:

not specified

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: other:

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

not specified

Skeletal malformations:

not specified

Visceral malformations:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Teratogenicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No treatment-related effects on any reproductive indices (for embryo/foetal toxicity).

Malformations:
- one vehicle control group foetus with unilateral testes
- one high-dose group foetus with acaudia (which also had absent sacral and lumbar vertebrae).

Skeletal variations:
- mainly of the sternebrae, skull, hyoid arch but without significant difference between groups or dose-relationship.

Dose descriptor:

NOAEL

Effect level:

300 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

26 females were pregnant at termination in all groups except at 30 mg/kg/day where all 27 were pregnant: all had viable foetuses.

Group mean data.

Embryo/foetal index	Vehicle controls	Test substance 30 mg/kg/day	Test substance 95 mg/kg/day	Test substance 300 mg/kg/day
Viable foetuses/dam	13.8 +/- 2.78	13.3 +/- 2.44	14.1 +/- 1.88	13.2 +/- 3.35
% Post-implantation loss	7.2	10.6	6.4	13.8
% Pre-implantation loss	6.8	6.4	6.1	4.4
Foetal sex distribution: % male	49.4	56.1	47.8	52.6
Foetal bodyweight   (by dam)	4.1 +/- 0.26	4.1 +/- 0.28	4.2 +/- 0.28	4.1 +/- 0.30

Conclusions:

In this study repeated dermal application of the tested analogue to rats produced local skin reactions in dams but no evidence of teratogenicity or embryo/foetal toxicity. Based on the close similarity of chemical structure and properties, it is predicted that the registered substance would give similar results in this assay.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

mouse

Quality of whole database:

Reliable (Klimisch 2) key study, supported by read-across from rat oral studies performed using a close chemical analogue.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Read-across from a reliable (Klimisch 1) study performed under GLP and using a standard test method (also supported by a rat oral study).

Additional information

In the key oral mouse study, repeated administration of the registered substance

In an oral rat study, repeated administration of the chemical analogue at 2.5x the NOAEL for subchronic oral toxicity produced no evident developmental toxicity.

In the dermal rat study, repeated application of a close chemical analogue of the registered substance at a level causing evident local skin reaction produced no evidence of teratogenic, embryotoxic or foetotoxic activity.

Justification for selection of Effect on developmental toxicity: via oral route:

No adverse effects seen in the key mouse developmental toxicity study using oral administration at dosages up to 300 mg/kg/day.

Justification for selection of Effect on developmental toxicity: via dermal route:

No adverse effects seen in the rat developmental toxicity study using dermal administration of a close chemical analogue at dosages up to 300 mg/kg/day.

Justification for classification or non-classification

Studies of developmental toxicity in the mouse and (using read-across) oral and dermal studies in the rat found no evidence of developmental toxicity. Consideration of all relevant data led to the conclusion that reproductive toxicity (fertility) testing is not necessary, as low toxicity to reproduction can be predicted. Accordingly, no classification in respect of reproductive toxicity is warranted.