3,3'-methylenebis[5-methyloxazolidine]

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 14, 2008 to March 30, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well performed Guideline study according to good scientific standards

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC 88/302; L 133/43 (1988)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Details on species / strain selection:

HanRcc (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories Ltd., 4414 Füllinsdorf, Switzerland
- Age at study initiation: (P) 7 wks
- Weight at study initiation: (P) Males: 192 to 229 g; Females: 135 to 177 g;
- Fasting period before study: no
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period:Under test conditions after health examination. Only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (12 hrs dark / 12 hrs light):

IN-LIFE DATES: From April 14, 2008 to August 19, 2008

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: 3,3’-methylen-bis-(5-methyloxazolidine) (MBO) was weighed into a glass beaker on a tared precision balance and the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration
period using a magnetic stirrer.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Cornoil was used to prepare a homogeneous suspension
- Concentration in vehicle: 0, 0.1, 0.3 and 0.9
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): Roth / 13890412

Details on mating procedure:

- M/F ratio per cage: 1.1
- Length of cohabitation: 14 days maximum
- Proof of pregnancy: [a) The daily vaginal smear was sperm positive, or b) A copulation plug was observed. Referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: No. If mating was not recorded during this additional pairing period of a maximum of 14 days, the female was sacrificed and the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.
- After successful mating each pregnant female was caged individually
- Any other deviations from standard protocol: None

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. In addition, samples of about 2 g of each concentration were taken from the middle to confirm stability (6 hours). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose
formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Dr. D. Flade (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.
The test item concentrations were determined by LC-MS developed at Harlan Laboratories.

Duration of treatment / exposure:

Pre-Pairing 70 days
Pairing 14 days maximum
Gestation approximately 21 days
Lactation 21 days
Treatment ended on day 20 post partum

Frequency of treatment:

Once daily

Details on study schedule:

- Selection of pups: All dams were allowed to give birth and rear their litters (F1 pups) up to day 21 post partum. Day 0 was designated as the day on which a female had delivered all her pups. The offspring was examined as soon as possible after completion of delivery for litter size, number of live and still births, and any gross anomalies. On day 4 post partum, the number of offspring was reduced to 8 per litter (when possible equally divided as to sex). On the day of weaning (day 21 post partum), the dams were separated from their litters.

Remarks:

Doses / Concentrations:
0, 5, 15, and 45 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

24 males/24 females per sex and dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous non-GLP compliant dose range finding toxicity study in Han Wistar Rats, Harlan Laboratories Study B50916, using dose levels of 30, 60 and 120 mg/kg/ day, resulting in a NOEL of 30 mg/kg/day.

- Rationale for animal assignment: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy).
Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Was recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION
Males: Weekly during pre-pairing and after pairing periods
Females: Weekly from treatment start to delivery and on days 1, 7 and 14 post partum.
No food consumption was recorded during the pairing period.

WATER CONSUMPTION No

Oestrous cyclicity (parental animals):

Determined in all femal parental animals

Sperm parameters (parental animals):

Parameters examined in all P male parental generations:
- Weights of seminal vesicles, with coagulating glands and their fluids (as one unit)
Epididymides (total weight and cauda separately)
Testes
Prostate

Histopathology from testis and epididymis

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (equally dividedas to sex as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, F1 pups were sacrificed on day 4 p.p., examined macroscopically for possible defects and preserved in neutral phosphate buffered 4% formaldehyde solution. All the remaining pups were sacrificed and examined macroscopically for defects after weaning. If indicated, skeletal development of the pups were examined by staining with alizarin red S after clearing in potassium hydroxide (a slightly modified technique of Dawson.
Dead young, except those excessively cannibalized, were autopsied and/or preserved in fixative for possible further examination.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals [at or shortly after weaning, P generation females and the remaining pups were sacrificed. P generation males were sacrificed when they were no longer needed for the assessment of reproductive effects..]
- Maternal animals: All surviving animals [females which lost their litter were sacrificed and necropsied together with the dams after weaning of the pups or if considered appropriate, at any time after litter loss. If birth did not occur on the expected date (day 21 p.c.), the female was sacrificed on day 25 p.c. and examined as described.]

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathology
(from Parental animals)

Gross lesions
Ovaries
Pituitary
Prostate
Seminal vesicles with coagulating gland
Testes with epididymides (in Bouin’s fixative)
Uterus and cervix
Vagina
Stomach
Slides of all organs and tissues (see list above) from all animals of the control and high dose group (group 1 and 4, respectively), and all gross lesions from all animals of all groups were processed, embedded in paraffin, cut at a nominal thickness of 2-4 micrometers, stained with hematoxylin & eosin (H&E) and examined by light microscope by the study pathologist. As histomorphologic changes were observed in the stomach of high dose animals of both sexes, examination of this organ was extended to the assigned animals of the mid- and low-dose groups


Organ weights (P)

Seminal vesicles, with coagulating glands and their fluids (as one unit)
Epididymides (total weight and cauda separately)
Testes
Prostate
Ovaries
Uterus (including cervix and vagina, excluding oviducts)
[Paired organs were weighed individually]

Postmortem examinations (offspring):

SACRIFICE
- F1 offspring
F1 pups selected for litter size standardization were sacrificed on day 4 p.p., examined macroscopically for possible defects and preserved in neutral phosphate buffered 4% formaldehyde solution. All the remaining pups were sacrificed and examined macroscopically for defects after weaning. If indicated, skeletal development of the pups were examined by staining with alizarin red S after clearing in potassium hydroxide (a slightly modified technique of Dawson).
Dead young, except those excessively cannibalized, were autopsied and/or preserved in fixative for possible further examination.

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:
• Means and standard deviations of various data were calculated and included in the report.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables can be dichotomized without loss of information

Reproductive indices:

Estrus Cycle
Duration of Gestation
Implantation Rate
Post-implantation Loss and Postnatal Loss Days 0 - 4 Post Partum

Offspring viability indices:

External Examination at First Litter Check and during Lactation
Sex Ratios
Litter Size at First Litter Check
Breeding Loss Days 5 - 21 Post Partum

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All animals survived until the scheduled necropsy.
No clinical signs or signs of discomfort were noted during the treatment period in any group in
males and females.
As an exception, one female in group 4 was lethargic, lost body weight and had diarrhea, ruffled
fur and a hunched posture on day 22 post coitum, indicating a dystocia. After giving birth (all
pups were cannibalized), no clinical sings were noted anymore.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males
In group 4, mean body weight gain was slightly although not statistically significantly reduced
during the pre-pairing period resulting in slightly reduced mean body weights. Mean body weights remained reduced until the end of the treatment period.
In groups 2 and 3, mean body weights were similar to the control group.

Females
Mean body weight and mean body weight gain were not affected by treatment with the test item in any group.
In groups 2 and 3, mean body weights were similar to the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males
In group 4, mean food consumption was slightly and occasionally statistically significantly
reduced during the pre-pairing period.
In groups 2 and 3, mean food consumption was similar to the control
group.

Females
In group 4, mean food consumption was slightly although not statistically significantly reduced
in the last week of the gestation period (-6.3% compared to the control group).
In groups 2 and 3, mean food consumption and mean was similar to the control
group.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

HISTOPATHOLOGY (PARENTAL ANIMALS)
Microscopically, MBO induced test item-related changes in the forestomach. They consisted of moderate to massive ulcerations of the forestomach in many male animals of group 4 as well as moderate to marked ulcerations in females of group 4. Minimal to slight vacuolar degeneration of the squamous epithelium was recorded only in group 4 males. Furthermore, there was minimal to moderate squamous hyperplasia along with different severity degrees of hyperkeratosis, submucosal inflammation and submucosal edema in animal of both sexes of group 4. In group 3, minimal to slight vacuolar degeneration as well as minimal degrees of squamous hyperplasia, hyperkeratosis, submucosal inflammation and edema were recorded in the forestomach of males but not in females. In addition, one male showed minimal focal erosion and minimal epithelial inflammatory infiltrates were observed in few males of group 3.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: 0, 5, 15, and 45 mg/kg bw/day

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
In group 4, there were indications for an undetected postnatal loss, which means pups were cannibalized directly after birth by its mother before their number and sex have been recorded. Combined total number of post implantation loss and number of postnatal loss between days 0 -4 were 47, 45, 48 and 66 pups in order of ascending dose levels. Since the number in groups 2 and 3 was similar than in the control group this finding was considered to be not test item-related.
In group 4, the high number of 66 lost pups and the high number of dead pups at first litter check were considered to be test item-related

Dose descriptor:

NOAEL

Effect level:

>= 5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

>= 15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: A systemic NO(A)EL for the parental animals was considered to be 15 mg/kg body weight/day, based on the reduced body weights at 45 mg/kg body weight/day.

Dose descriptor:

NOAEL

Effect level:

>= 15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The NOAEL for reproduction/ developmental toxicity was considered to be 15 mg/kg/day, based on the increased incidence of the post implantation and postnatal loss at 45 mg/kg body weight/day.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

During external examination at first litter check no test item-related findings were noted. The sex ratio was similar in all groups.

Pup Weights to Weaning (to Day 21 Post Partum)
Mean pup weights on day 1 post partum and during the lactation period were not affected by treatment with the test item.

Macroscopical Findings
At necropsy of pups, there were no abnormal findings.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 45 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects were noted in the F1 progeny during the weaning phase and at necropsy.

Reproductive effects observed:

not specified

Table 1                           Table for animal assignment for mating
		Number of animals
		Controls	Low Dose	Medium Dose	High Dose
Parents (P)	m	24	24	24	24
	f	24	24	24	24
F1	Litters (total)	21	22	21	22

Table 2                           Table for reproductive toxicity study (Only relevant findings presented)
Parameter			control		low dose		medium dose		High dose
		Generation	m	f	m	f	m	f	m	f
Mortality	incidence	P	0	0	0	0	0	0	0	0
Food consumption(pre-pairing period, days 1-70)	% of control	P			-1.5	+1.5	-1%	+2.9	-5.8	+1.5
Food consumption(females, last gestational week, day 14-21)	% of control	P				+5.2		+2.9		-6.3
Differences in mean body weight gain(pre-pairing period, from day 1 to 70)	% of control	P	+108	+55	+103	+54	+107	+56	+101	+57
Clinical Observations	Incidence	P	n.s.f.	n.s.f.	n.s.f.	n.s.f.	n.s.f.	n.s.f.	n.s.f.	1#
Pathology		P
Macroscopic findings (Stomach)		P
Cratiform retractions	Incidence, (%)	P	0	0	0	0	0	0	9 (38)	1 (4)
Isolated cratiform retractions	Incidence, (%)	P	0	0	0	0	0	0	3 (13)	1 (4)
Nodules	Incidence, (%)	P	0	0	0	0	0	0	5 (21)	0
Several cratiform retractions	Incidence, (%)	P	0	0	0	0	0	0	1 (4)	0

n.s.f.: no specific findings

#: one high-dosed female showed lethargia, diarrhea and dystocia and had cannibalized its pups

 

Parameter			control		low dose		medium dose		High dose
		Genera­tion	m	f	m	f	m	f	m	f
Histopathologic examination (Forestomach)
No. animals examined		P	24	24	24	24	24	24	24	24
Ulcerations	Incidence	P	0	0	0	0	0	0	19	4
Vacuolar degeneration	Incidence	P	0	0	0	0	4	0	5	0
Squamous hyperplasia	Incidence	P	0	0	0	0	5	0	10	5
Hyperkeratosis	Incidence	P	0	0	1	0	5		18	7
Submucosal inflam.	Incidence	P	0	0	0	0	2	0	4	2
Reproductive Performance
Implantations	Group means	P		12.8		13.0		13.3		13.1
Post implanation loss	% of implantations	P		7.5		12.3		14.7		16.3
	Litters affected	F1		12		12		14		10
	Total	F1		20		35*		41**		47**
	Mean	F1		1.0		1.6		2.0		2.1
	N	F1		21		22		21		22
Post natal loss (days 0-4 p.p.)	% of living pups	F1		10.9		4.0		2.9		7.9
	Litters affected	F1		4		8		3		5
	Total	F1		27		10**		7**		19
	Mean	F1		1.3		0.5		0.3		0.9
	N	F1		21		22		21		22
Dead pups at first litter check	Litters affected	F1		3		3		3		3
	Total	F1		3		2		12		24
	Mean	F1		0.1		0.1		0.6		1.1
	N	F1		21		22		21		22

 

* or **: Fisher’s Exact test at 5% or 1% level

Conclusions:

Based on histopathological test-item related toxicological relevant effects in the forestomach in male and female rats administered at 15 and 45 mg/kg, a general local No-Observed-Adverse-Effect-Level (NOAEL) for the parental animals was considered to be 5 mg/kg body weight/day. A systemic NO(A)EL for the parental animals was considered to be 15 mg/kg body weight/day, based on the reduced body weights at 45 mg/kg body weight/day. The NOAEL for reproduction/ developmental toxicity was considered to be 15 mg/kg/day, based on the increased incidence of the post implantation and postnatal loss at 45 mg/kg body weight/day. No effects were noted in the F1 progeny during the weaning phase and at necropsy.

Executive summary:

One generation reproductive toxicity study according to OECD Guideline 415.

MBO was administered in corn oil as vehicle at dosages of 5, 15, and 45 mg/kg body weight/day.

In group 2 (5 mg/kg bw), no test item-related findings were noted.

In group 3 (15 mg/kg bw), histopathological changes were recorded in the forestomach of males but not in females.

In group 4 (45 mg/kg bw) in males, mean food consumption and mean body weight gain were slightly reduced during the pre-pairing period, resulting in slightly reduced mean body weights. In females, only mean food consumption was slightly although not statistically significantly reduced in the last week of the gestation period.

The sum of the post implantation loss, the number of dead pups at first litter check and the postnatal loss between days 0 - 4 was increased.

Macroscopically, test item-related crateriform retractions and/or nodules in the forestomach mucosa of many males and of few females were recorded. These findings were histopathologically confirmed.

The NOAEL (parental) for reproduction was 15 mg/kg bw. No effectes were detected in the F1 progeny.

Effect on fertility: via oral route

Dose descriptor:

NOAEL

15 mg/kg bw/day

Additional information

Based on histopathological test-item related toxicological relevant effects in the forestomach in male and female rats administered at 15 and 45 mg/kg, a general local No-Observed-Adverse-Effect-Level (NOAEL) for the parental animals was considered to be 5 mg/kg body weight/day. A systemic NO(A)EL for the parental animals was considered to be 15 mg/kg body weight/day, based on the reduced body weights at 45 mg/kg body weight/day. The NOAEL for reproduction/ developmental toxicity was considered to be 15 mg/kg/day, based on the increased incidence of the post implantation and postnatal loss at 45 mg/kg body weight/day. No effects were noted in the F1 progeny during the weaning phase and at necropsy.

Short description of key information:
Effects on male and female reproductive performance was investigated in a one-generation reproduction toxicity study according to OECD guideline 415 (Senn 2009).

MBO was administered in corn oil as vehicle at dosages of 5, 15, and 45 mg/kg body weight/day.
In group 2 (5 mg/kg bw), no test item-related findings were noted.
In group 3 (15 mg/kg bw), histopathological changes were recorded in the forestomach of males but not in females.
In group 4 (45 mg/kg bw) in males, mean food consumption and mean body weight gain were slightly reduced during the pre-pairing period, resulting in slightly reduced mean body weights. In females, only mean food consumption was slightly although not statistically significantly reduced in the last week of the gestation period.
The sum of the post implantation loss, the number of dead pups at first litter check and the postnatal loss between days 0 - 4 was increased.

Macroscopically, test item-related crateriform retractions and/or nodules in the forestomach mucosa of many males and of few females were recorded. These findings were histopathologically confirmed.
The NOAEL (parental) for reproduction was 15 mg/kg bw. No effectes were detected in the F1 progeny.

Effects on developmental toxicity

Description of key information

In a prenatal developmental toxicity study in rabbits according to OECD guideline 414 (Cordts 2008) rabbits were gavaged with 0, 5, 45, 90, 135 mg/kg bw/day corresponding to a concentration of 0, 0.25, 2.25, 4.5, 6.75% in corn oil. A dose of 135 mg/kg bw/day resulted in severe maternal toxicity like a decrease in body weight, increased mortality and abortions. Necropsy revealed local lesions in the stomach of dams and an increased incidence in dilatation of the renal pelvis. The authors of the study suggested a NOEL for maternal toxicity at 90 mg/kg bw/day. However, there is some evidence that at least an increased incidence of lesions in the stomach occurred also at 45 mg/kg bw. Developmental toxicity like an increased number of early and late resorptions, a decreased number of foetuses, an increase in post-implantation loss and mortality of foetuses was only observed at 135 mg/kg bw/day, a dose which resulted also in severe maternal toxicity. No increase in the incidence of retardations, variations or malformations was detected in any treatment group.

Link to relevant study records

Reference

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)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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)

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

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)

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).

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).

Dose descriptor:

NOEL

Effect level:

>= 90 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

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.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

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

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.

Effect on developmental toxicity: via oral route

Dose descriptor:

NOAEL

15 mg/kg bw/day

Additional information

The substance induced developmental effects like decreased foetal weight but no teratogenicity at dose levels resulting in severe maternal toxicity predominantly via local effects on the gastro-intestinal tract after oral exposure. Similarly, formaldehyde has developmental effects but only at dose levels with severe local maternal toxicity after inhalation or oral exposure. There is also no evidence for teratogenicity of formaldehyde. No data are available on 2-hydroxypropylamine. However, in comparison to the other components the data on repeated dose toxicity of 2-hydroxypropylamine (although of limited validity) suggested that toxic effects of 2-hydroxypropylamine occurred at much higher dose levels.

In summary, there is no evidence for adverse effects of the substance on embryo and foetal development at dose levels inducing no local maternal toxicity.

The implementation of a teratogenicity study in a 2nd species is scientifically unjustified because also no teratogenic effects are expected due to concentration dependent local effects.

Justification for classification or non-classification

In summary, there is no evidence for adverse effects of the substance on fertility or embryo and foetal development at dose levels inducing no local maternal toxicity. Therefore, the substance is not subject of classification.

The implementation of a teratogenicity study in a 2nd species is scientifically unjustified because also no teratogenic effects are expected due to concentration dependent local effects.