3,3'-methylenebis[5-methyloxazolidine]

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3,3’-methylen-bis-(5-methyloxazolidine) (MBO)

IUPAC Name:

3,3’-methylen-bis-(5-methyloxazolidine) (MBO)

Details on test material:

- Name of test material (as cited in study report): 3,3’-methylen-bis-(5-methyloxazolidine) (MBO) (Grotan OX)
- Substance type: Formaldehyde releaser
- Physical state: Liquid, colorless to light yellow
- Analytical purity: 90 - 100%
- Composition of test material, percentage of components:
- Isomers composition:
- Purity test date: Oct 22, 2007
- Lot/batch No.: 0001129974
- Stability under test conditions: The application formulations investigated during the study were found to comprise MBO in the range of 61.0% to 84.5%. Due to a challenging development of a suitable analytical method, for which the validation took more time than planned, the samples were stored for more than one year. Certain degradation of the formulated test item cannot be completely excluded due to this long storage time of samples. Therefore, the NOEL was not corrected according to the measured recovery concentrations.
- Storage condition of test material: Dry and at room temperature (20 ± 5 °C)

Test animals

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

Administration / exposure

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.

Doses / concentrations

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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

Effect levels (P0)

1

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'

2

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.

3

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.

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.