3,3'-methylenebis[5-methyloxazolidine]

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2005 (Study Plan) to April 02, 2008 (Final Report)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study, well performed according to good scientific standards. On a few test days the relative humidity was outside the range 40-70% (minor restriction)

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2004

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,3'-methylenebis[5-methyloxazolidine]

EC Number:

266-235-8

EC Name:

3,3'-methylenebis[5-methyloxazolidine]

Cas Number:

66204-44-2

Molecular formula:

C9H18N2O2

IUPAC Name:

3,3'-methylenebis[5-methyloxazolidine]

Details on test material:

- Name of test material (as cited in study report): N,N'-Methylen-bis-(5-methyloxazolidine) (MBO) (GrotaMar 71)
- Substance type: Formaldehyde releaser
- Physical state: Almost clear, colourless liquid
- Analytical purity: 99%
- Composition of test material, percentage of components: Reaction product from paraformaldehyde and 2 hydroxypropylamine (ratio of 3:2)
- Purity test date: 29. Nov 2005
- Lot/batch No.: 1094394
- Expiration date of the lot/batch: June 2007
- Stability under test conditions: Stability and homogeneity of the test substance in corn oil has been shown
- Storage condition of test material: At room temperature

Test animals

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: LPT, Branch Facility Löhndorf, 24601 Löhndorf, Germany
- Age at study initiation: 4-5 months
- Weight at study initiation: 2.33-3.24 kg body weight
- Fasting period before study: no
- Housing: Breeding cages with wire floors
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: In-house bred, their state of health was checked prior to the start of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C ± 3°C
- Humidity (%): 55% ± 15%
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From December 12, 2005 (first administration) to February 16, 2006 (termination of inlife phase)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was diluted in the vehicle to the appropriate concentrations


VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is instable in water, formaldehyde will be deceased off. Hence, at
the request of the sponsor, corn oil was employed as vehicle, however, as little as possible.
- Concentration in vehicle: 0, 0.25, 2.25, 4.5, 6.75%
- Amount of vehicle (if gavage): 2 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test substance in corn oil has been measured in an analytical study (Preiß, 2006). Samples collected immediately after preparation of the formulation as well as 8 h after storage were analysed. Homogeneity was examined in samples collected at the start of administration, during administration and before treatment of the last animal. Stability and homogeneity of the test substance in corn oil has been shown.

Details on mating procedure:

- Impregnation procedure: Monogamous mating
- M/F ratio per cage: 1/1
- Length of cohabitation: Until successful copulation was observed
- Further matings after unsuccessful attempts: Rabbits lacking signs of copulation were excluded from the analysis
and replaced by additional spare animals.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: Successful copulation was ascertained by observation (considered as day 0 of pregnancy).

Duration of treatment / exposure:

Day 6 to 28 of pregnancy

Frequency of treatment:

One daily oral gave treatment

Duration of test:

Until Day 28 of pregnancy. Sacrifice on Day 29

No. of animals per sex per dose:

24 animals per group at initiation

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a preliminary study 2 pregnant rabbits per dose received 20, 60, 180 mg/kg bw/day on day 6-28 of pregnancy in 2 ml/kg bw corn oil. NOEL for effects on foetuses 60 mg/kg bw. Maternal NOEL <20 mg/kg bw/day (slight gastric lesions); lethal effects at 180 mg/kg bw.
- Rationale for animal assignment (if not random): random

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Viability checked twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily data were recorded. Dated and signed records of appearance, change and disappearance of clinical
signs were maintained on clinical history sheets for individual animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Determined daily (day 0-29), always at the same time of the day

FOOD CONSUMPTION Yes
- Daily food consumption recorded and relative food consumption calculated (related to body weight)

WATER CONSUMPTION
- Daily monitoring by visual appraisal of the drinking water consumption was maintained
throughout the study

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: In order to check for possible drug effects, a dissection with macroscopic examination
of the internal organs of the dams was carried out on the day of scheduled laparotomy or on the day on which the animals were found dead.


OTHER:
The kidneys (2) of all dams were preserved in 10% buffered formalin for possible future histopathological examination.
In case of macroscopical findings, the affected maternal tissues were preserved in 10% buffered formalin for possible future histopathological examinations.

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
- Other: Weight of placentae

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]: The thorax and peritoneal cavity (without damage to ribs and sternum)
were opened; location, size and condition of the internal organs were determined and examined for abnormalities of soft tissue
- Skeletal examinations: Yes: [all per litter form the eviscerated fetuses (intact and headless bodies)]
- Head examinations: Yes: [half per litter]

- Other: Litter size, no. of dead & alive foetuses, no of placentae, foetal weight measured; sex ratio; and viability after a 6 h and a 24 h stay in the incubator at 34°C determined; foetuses examined for external malformations; thereafter foetuses sacrificed (ether) and dissected;

Statistics:

For all numerical values, homogeneity of variances was tested using the BARTLETT chi-square test.
When the variances were homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group.

In case of heterogeneity of variances, the STUDENT's t-test was carried out, limit of significance was p ≤ 0.01.

For the comparison of classification measurements (for example malformation-, resorption-, retardation- and variation rate) the FISHER's exact test (n < 100)
or chi2-test with YATES' correction for continuity (n ≥ 100) (p ≤ 0.05 and p ≤ 0.01) was employed.

Indices:

Malformation rate [%]
Variation rate [%]
Retardation rate [%]
Pre-implantation loss [%]
Post-implantation loss [%]

Historical control data:

Yes: Background data available
(summarized results of the 30 last embryotoxicity studies in rabbits (Himalayan) performed at LPT in the years 2000 - 2005)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

≤ 90 mg/kg bw/day
these dose levels caused no test substance related clinical effects

Mortality:

mortality observed, treatment-related

Description (incidence):

≤ 90 mg/kg bw/day
these dose levels caused no test substance related mortality.

135 mg/kg bw/day
increased mortality (10 pregnant rabbits between gestation day 9
and 23

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

≤ 90 mg/kg bw/day
No effects

135 mg/kg bw/day
body weight reduced (max. 9%); the body weight change was
significantly reduced at gestation day 9-12, 18-21, and 27-29.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

≤ 90 mg/kg bw/day
No effects

135 mg/kg bw/day
Treatment related effects on absolute and relative food
consumption; significant at gestation day 11-14 and 28.

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

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

135 mg/kg bw/day
decrease of gravid uterus weight

Gross pathological findings:

no effects observed

Description (incidence and severity):

≤ 90 mg/kg bw/day:
Soft faeces and gastric lesions were noted in
several animals including controls; this is presumably caused by
the vehicle corn oil (possible confounding factor).

135 mg/kg bw/day
severe gastro-intestinal lesions in prematurely deceased dams
(n=10), in dams with abortion (n=4) and in surviving dams.
Increased incidence of dilatation of the renal pelvis

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

≤ 90 mg/kg bw/day
No effects

135 mg/kg bw/day
severe gastro-intestinal lesions in prematurely deceased dams
(n=10), Necropsy revealed changes of the kidney (dilatation of renal pelvis).

Maternal developmental toxicity

Description (incidence and severity):

≤ 90 mg/kg bw/day
The abortions in controls and treated groups are within the normal variability
(1-2 per group).These
findings were considered to be within the spontaneous range.

135 mg/kg bw/day
increased abortions

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

≤ 90 mg/kg bw/day
Total post implantation loss was noted in one dam at 5 and 90 mg/kg bw/day. These
findings were considered to be within the spontaneous range.

135 mg/kg bw/day
post-implantation loss (n=3) (52.6% versus 13.7% in
control).

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

≤ 90 mg/kg bw/day
no effect

Early or late resorptions:

no effects observed

Description (incidence and severity):

≤ 90 mg/kg bw/day
no effect

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

≤ 90 mg/kg bw/day
no effect

135 mg/kg bw/day
significant increase

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

≤ 90 mg/kg bw/day
no effect

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations
No test substance related findings were recorded at external
examination of foetuses. External malformations were detected in
all groups including control excluding high dose group (but only
5 dams evaluated) and were considered to be of spontaneous
nature. No test substance related macroscopic visible variations
were detected.
Skeletal examination of the foetuses revealed no test substance
related increase in variations, retardations or malformations in
any group. The observed effects were within the spontaneous
range.

Details on maternal toxic effects:

≤ 90 mg/kg bw/day
No developmental effects were detected with respect to the
number of corpora lutea (determined values: number per dam,
absolute number per group, mean per group), implantations
(number per dam, distribution in uterine horns, absolute number
per group, mean per group), resorptions (number per dam,
distribution in uterine horns, absolute number per group, mean
per group, mean % per group, early resorptions, late resorptions),
weight of placenta (individual data per foetus, mean per litter,
mean per group, litter mean per group, litter mean per sex and
group), weight of foetuses (individual data per foetus, mean per
litter, mean per sex and litter, litter mean per group, litter mean
per sex and group), foetuses (number of alive or dead per dam,
number per sex and dam, distribution in uterine horns, absolute
number alive per group, mean number alive per group, mean%
alive per group, mean % per sex and group) when compared to
the control. Abortions were found in 1-2 dams per group
including the control (n=2 abortions). Total post implantation
loss was noted in one dam at 5 and 90 mg/kg bw/day. All these
findings were considered to be within the spontaneous range.
135 mg/kg bw/day
Due to the high mortality rate (n=10 dams) and abortions (n=4)
and/or post-implantation lost (n=3) only 5 litters could be
evaluated. The number of early and late resorptions was
increased (p≤0.01), the number of foetuses decreased (p≤0.01)
and the post-implantation loss increased (52.6% versus 13.7% in
control). The mortality of foetuses during the 6-h incubator stay
was significantly increased (9/24; p<0.01).

Effect levels (maternal animals)

Dose descriptor:

NOEL

Effect level:

>= 90 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
At 135 mg/kg b.w./day, a total post-implantation was noted in 3 of 8 examined dams. The number of early and total resorptions was significantly increased, the number of fetuses accordingly decreased.
At 5, 45, 90 or 135 mg/kg b.w./day, no test item-related malformations or variations were noted during external or skeletal examination of the fetuses
(according to DAWSON) and soft tissue examination of the fetal heads (according to WILSON).
Skeletal examination (according to DAWSON) revealed no test item-related retardations at any tested dose level.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

>= 90 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Macroscopic findings in the stomach and the kidney of dams at necropsy (at gestation day 29 & prematurely deceased rabbits)

Finding	Control n=23#	5 mg/kg bw n=24#	45 mg/kg bw n=23#	90 mg/kg bw n=21#	135 mg/kg bw n=22#
Stomach
Detachment and/or reddening of mucosa	1#	2#	1#	0	10#
Reddened stomach	0	0	3	3#	10#
Ulcer(s)	0	2#	1	0	4#
Haemorrhagic/dark/ reddish/black foci	0	0	1	4#	8#
Distensions	0	0	0	0	3
Any lesion (from individual data, Table A7)	1/23	2/24	6/23	7/21	22/22
Duodenum
reddened	0	0	0	0	2#
Kidney
Dilatation of renal pelvis	1	4#	4#	4	12#
Coarse structure of cortex	0	0	2	0	2#
Very soft tissue	0	0	0	0	1
#: (including) prematurely deceased dams

Developmental toxicity

Finding	Control n=20	5 mg/kg bw n=21	45 mg/kg bw, n=20	90 mg/kg bw, n=17	135 mg/kg bw, n=8
Corpora lutea total	158	166	149	131	63
Corpora lutea per dam	7.9	7.9	7.5	7.7	7.9
Total implantation sites	135	133	128	104	51
Implantation sites/dam	6.8	6.3	6.4	6.1	6.4
Total resorptions	15	15	12	9	27**
Resorptions/dam	0.8	0.7	0.6	0.5	3.4
Total early resorptions	11	15	11	8	26**
Early resorptions/dam	0.6	0.7	0.6	0.5	3.3
Total late resorptions	4	0*	1	1	1
Late resorptions/dam	0.2	0.0	0.1	0.1	0.1
Mean% pre-implantation loss	14.8	18.6	16.7	20.0	18.0
Mean% post-implantation loss	13.7	10.3	9.5	10.1	52.5
Total live foetuses	120 (n=20)	118 (n=20)	116 (n=20)	95 (n=16)	24** (n=5)
Live foetuses per dam	6.0	5.9	5.8	5.9	4.8
Total dead foetuses at laparatomy	0	0	0	0	0
*: p≤0.05; **: p≤0.01

Applicant's summary and conclusion

Conclusions:

The test substance has no teratogenic properties. The observed developmental effects occurred only at dose levels inducing severe maternal toxicity.

Executive summary:

Study according to OECD guideline 414. Prenatal developmental toxicity study of the test substance in rabbits by oral administration. 24 pregnant rabbits per dose were gavaged with 0, 5, 45, 90, 135 mg/kg bw/day (concentration: 0, 0.25, 2.25, 4.5, 6.75% in corn oil) at gestation day 6-28.

Maternal toxicity: At 135 mg/kg bw/day a decrease in body weight, increased mortality and abortions indicated clear maternal toxicity. Necropsy revealed local lesions in the stomach and an increased incidence in dilatation of the renal pelvis.
No effects were found at 90 mg/kg bw/day. The effects in the stomach (see Table above) were related by the authors to the vehicle corn oil. The authors concluded that no test substance related effects were found at 90 mg/kg bw/day.

Developmental toxicity: No developmental effects were detected at 90 mg/kg bw/day. At the high dose level the number of early and late resorptions was increased, the number of foetuses decreased, the post-implantation loss and the mortality of foetuses during the 6-h incubator stay increased. No increase in the incidence of retardations, variations or malformations was detected in any treatment group.

In conclusion, the test item N,N'-Methylen-bis-(5-methyloxazolidine) possessed no teratogenic properties. Embryotoxic properties were noted only at severe maternal toxicity at 135 mg/kg b. w./day in form of an increased resorption rate.