3,3'-methylenebis[5-methyloxazolidine]

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sept.24, 2001 to Dec. 17, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

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): 3,3'-methylenebis[5-methyl-oxazolidine]
- Substance type: Formaldehyde releaser
- Physical state: Clear, colourless liquid
- Analytical purity: content of formaldehyde 42.28%,
- Composition of test material, percentage of components: Reaction product from paraformaldehyde and 2 hydroxypropylamine (ratio of 3:2)
- Purity test date:
- Lot/batch No.: 24773
- Expiration date of the lot/batch: No data
-- Stability under test conditions: Stability was proven analytically
- Storage condition of test material: At room temperatur in tightly closed original container

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, D-97633 Sulzfeld
- Age at study initiation: At 1st dose: m 32 days, f 34 days
- Weight at study initiation: m 82-92 g and f 80-90 g upon receipt.
- Fasting period before study: no
- Housing: Single in Macrolon Type III cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3
- Humidity (%): 5 +- 15
- Photoperiod (12 hrs dark / 12 hrs light):

IN-LIFE DATES: From January 10, 2002 to April 10, 2002

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

The test item was suspended in corn oil to the appropriate dose levels

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item forms a homogeneous suspension in corn oil
- Concentration in vehicle: 0, 4, 12, 36/24 mg/mL (0, 0.4, 1.2, 3.6/2.4%)
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration and stability of the test substance in dose solutions were tested in 3 samples per dose level. 0, 8, or 24 h after preparation the analytical concentration was max. 6.1% below the nominal concentration of the test substance at 0 h.
Homogeneity of the test substance during application period was tested on three samples per dose level; the variation was < 2%.
Samples at termination (last application to the last animal of each group) revealed analytical values resulting in 94.6-96.2% of nominal values.
Accuracy, precision, and sensitivity of the analytical method have been shown.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once daily, 7 days per week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 20, 60, 180/120 mg/kg bw; high dose level reduced to 120 mg/kg bw at the end of week 12 due to increased mortality.
Basis:
actual ingested

No. of animals per sex per dose:

10 m and 10 f per dose level

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected after a preliminary 28-Day dose range finding study (LPT Report 14814/01, dated January 09, 2002).
- Rationale for selecting satellite groups: No satellite groups included

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily each rat was observed for clinical signs, additionally immediately after each oral application. Mortality was checked twice daily and premortal symptoms recorded.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made in all animals once a week starting before the 1st application in a standard arena and included changes in skin, mucous membranes, autonomic activity, gait, posture, response to handling, as well the presence of abnormal secretions, abnormal movements or behaviour. At week 13 these observations were performed prior to any laboratory test.

BODY WEIGHT: Yes
- Time schedule for examinations: Measured once weekly starting day 0.

FOOD CONSUMPTION :
Calculated for each treatment week by the total amount of food given to and left by each rat in this week.

FOOD EFFICIANCY
No data

WATER CONSUMPTION
- Time schedule for examinations: Monitored daily.

OPHTHALMOSCOPIC EXAMINATION: Yes
Complete examination performed before exposure starts and prior to termination in all groups including recovery groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At day 90
- Anaesthetic used for blood collection: Yes :light ether anesthesia
- Animals fasted: Yes, overnight prior to sampling
- How many animals: All surviving animals per group and sex
- Parameters checked: Haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, activated partial thromboplastin time, thromboplastin time.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At day 90
- Animals fasted: Yes, overnight prior to sampling
- How many animals: All surviving animals per group and sex
- Parameters checked: Sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein, albumin, alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (aP).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At the end of the exposure period (>= week 11) functional observations were recorded including sensory reactivity to different types of stimuli (auditory, visual, proprioreceptive), assessment of grip strength and motor activity.
- Dose groups that were examined: all

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Necropsy of rats in all 4 groups performed.

HISTOPATHOLOGY: Yes
All organs which are listed in the OECD guideline 408 were fixed in
formalin. Histopathological examinations of these organs were done in
tissues of controls and animals of the high dose group including animals
which died during the exposure period.
Due to substance related changes also stomach and bone marrow of low
and mid dose rats were examined histopathologically

Other examinations:

Organ weights: The organ weights of liver, kidneys, adrenals, testes, epididymis, uterus, ovaries, thymus, spleen, brain, heart were determined before fixation. Same procedure with animals which died during exposure period but these data were not included in mean value comparison.

Statistics:

Suitable statistical methods were used and the level of significance was p<0.01 except in histopathology (p<0.05; Fisher exact test).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No treatment related effects were observed in the low and mid dose
groups. In the high dose group all males and females showed longlasting
piloerection from day 35 onwards. Ataxia was noted in one
female.
Functional observation battery
No treatment related effects were recorded in low and mid dose groups
at week 13. Reduced pupil size was detected in 3/7 male and 5/5 female
survivors. Further parameters were within the normal range.

Mortality:

mortality observed, treatment-related

Description (incidence):

No mortality in low and mid dose groups. High dose group: 3 males
died between day 49 and 75 and 5 females between day 49 and 79.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No effect on body weight gain was detected in the low and mid dose
group. From test week 5 onwards the body weight of males in the high
dose group was decreased. The difference to controls was -10 to -21%
(p<0.01). Less decreased body weights were measured in females. The
difference to controls was -6 to -18% (p<0.01 at week 10).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Transient effects on food consumption were measured in all treatment
groups. Significant decreases as well as increases were observed. No
dose dependency was detected. It was concluded by the authors that the
food consumption was not influenced by the test substance treatment.

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

by visual appraisal

Ophthalmological findings:

effects observed, non-treatment-related

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance related changes were presented in the Table below. No
effects were recorded at 20 mg/kg bw. In males of the mid dose group
an increase in leucocyte counts was demonstrated as well as a decrease
in activated partial thromboplastin time (aPTT) (but no significant effect
at the high dose level, no dose dependency). The thromboplastin time
(TPT) was decreased in males of all three dose groups but this effect
was not considered to be treatment related since the concurrent control
value was high in comparison to historical controls (data not shown by
the authors).
Males and females of the high dose group revealed an increase in the
incidence of neutrophilic granulocytes and a decrease in lymphocytes.
Effects on leucocyte counts and alteration in differential blood count are
suggested by the authors to be treatment related and a consequence of
the lesions detected in the stomach.

Clinical biochemistry findings:

effects observed, treatment-related

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

No effects on relative or absolute organ weights were detected in the
low and mid dose groups.
Survivors at 180/120 mg/kg bw: Significant increase (p<0.01) in relative
organ weight was measured for the following organs: brain (+20% in
males and +17% in females), gonads (+23% [left] and +18% [right] in
males), and liver (+24% in females).
The absolute organ weights in females were not altered but in males the
absolute weights of the kidney (left -20%, right -23%), spleen (-23%),
thymus (-35%), epididymis (left -21%, right -17%) were decreased (no
data on statistical significance).
The toxicological relevance of these effects was not discussed by the
authors. However, it is suggested that the increase in relative weights of
brain and gonads are related to the decrease in body weight and/or to
increased metabolism concerning the liver. Furthermore, no
histopathological changes were noted. The decreases of the absolute
organ weights in males are also of low toxicological relevance since the
body weight also decreases.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

20 mg/kg bw: No treatment related effects were recorded in the low
dose group.
60 mg/kg bw: In females no pathological alterations were reported. In 5
out of 10 males greenish discolorations/nodules were observed in the
stomach.
180/120 mg/kg bw: Greenish, brown-blackish or reddish discolorations/
nodules in the stomach of 8/10 males and 6/10 females. Inflated,
enlarged, or reddish discoloured intestinal tract was detected in 2/10
males and 3/10 females. Reduced size of the thymus (1/10 males and
1/10 females) and spleen (1/10 males and 1/10 females) were reported.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

20 mg/kg bw: The treatment did not induce any histopathological
alterations in the 2 organs examined in this group (stomach and bone
marrow).
60 mg/kg bw: Local effects in the stomach (see Table below) and
granulocytopoesis in bone marrow (inflammatory response) were
observed. Only these 2 organs were investigated.
180/120 mg/kg bw: Treatment related histopathological effects seen only in the stomach and bone marrow (see Table below). Further
changes in various organs were within the normal control range for
animals of this strain and age.
Authors comment: chronic ulcerative gastritis and peritonitis and/or
hyperplastic-hyperceratototic epithelial reactions with bacterial infection
especially of the proventricular part of the stomach in 6 males and 5
females of the mid dose group and all males and 9 females of the high
dose group.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

>= 20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

2

Dose descriptor:

LOAEL

Effect level:

>= 60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: local effects to the stomach

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Altered parameters in haematology

	Dose in mg/kg bw
Parameter	0	20	60	180/120
	Males
Leucocyte counts (10^6/ml)	10.3+-2.15	12.8+-1.58	15.5+-3.01*	11.1+-3.23
aPTT (sec)	12.6+-1.09	11.7+-0.91	10.8+-1.39*	11.2+-1.64
TPT (sec)	9.3+-0.39	7.9+-0.48*	8.0+-0.68*	7.8+-0.21*
Neutrophilic granulocytes (%)	16.4	12.7	19.7	22.6
Lymphocytes (%)	79.0	82.9	75.7	73.7
	Females
Neutrophilic granulocytes (%)	11.3	9.5	13.7	33.3
Lymphocytes (%)	84.7	86.9	82.0	62.6
*: significant at p<0.01; mean ± standard deviation

Table 2: Altered parameters in clinical chemistry of female rats

	Dose in mg/kg bw
Parameter	0	20	60	180/120
Albumin (mg/ml)	39.3+-2.23	40.4+-3.21	39.9+-3.52	34.3+-4.01*
Protein (mg/ml)	75.4+-3.5	77.6+-4.6	76.9+-3.7	68.4+-8.4*
*: significant at p<0.01; mean ± standard deviation

Table 3: Treatment related incidences of histopathological alterations
Data on terminal sacrifice reported

	Dose in mg/kg bw
Organ effect	0 males//females	20 males//females	60 males//females	180/120 males//females
Stomach Glandular ectasia	6/10 // 2/10	3/10 // 2/10	3/10 // 3/10	0/7* // 0/5
Submucosal lymphocytic hyperplasia	0/10 // 0/10	0/10 // 0/10	0/10 // 0/10	3/7 // 3/5*
Subepithelial mixed cell infiltration	0/10 // 0/10	0/10 // 0/10	1/10 // 5/10*	0/7 // 0/5
Epithelial hyperplasia	0/10 // 0/10	0/10 // 0/10	1/10 // 4/10	0/7 // 0/5
Chronic ulcerative gastritis & peritonitis	0/10 // 0/10	0/10 // 0/10	5/10* // 4/10	7/7** // 5/5**
Bone marrow  increased granu-  locytopoesis	0/10 // 0/10	0/10 // 0/10	4/10 // 1/10	4/7* // 1/5
*: significant at p<0.05; **: at p<0.001;

Applicant's summary and conclusion

Conclusions:

At dose level of ≥ 60 mg/kg bw and a concentration of ≥ 12 mg/mL in corn oil the test substance induced local effects in the stomach after repeated administration via gavage (LOAEL). Presumably higher LOAELs can be expected if the test substance is applied via drinking water or diet and not via gavage (bolus effect).
No effects were observed at a daily dose of 20 mg/kg bw. (NOAEL)

Executive summary:

The study was conducted according to OECD guideline 408 (adopted21^stSept. 1998). Ten male and 10 female rats per dose level were gavaged once daily, 7 days per week for 90 days with 0, 20, 60, 180/120 mg/kg bw (concentration: 0, 0.4, 1.2, 3.6/2.4% in corn oil).

The test substance induced local effects in the stomach at a dose level of ≥ 60 mg/kg bw. Other reported effects like piloerection and reduced body weight gain in the high dose groups and alteration in haematology like increased leucocyte counts and shift in the differential blood count at mid and high dose level are considered to be a consequence of the chronic ulcerative gastritis & peritonitis. The reduced pupil size detected in males and females and altered clinical chemistry parameters in females of the high dose group are recognized but the toxicological relevance is unclear.