1,3,5-trioxane

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline with acceptable restrictions, incomplete reporting (e.g. organ weigts)

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1983

Report date:

1983

Reference 2

Reference Type:

secondary source

Title:

1,3,5-TRIOXANE, CAS Number 110-88-3, USEPA HPV Challenge Program Submission

Author:

Trioxane Manufacturers Consortium Members, BASF Performance Copolymers, LLC (Formerly Ultraform Company) and Ticona

Year:

2000

Bibliographic source:

USEPA HPV Submission

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,3,5-trioxane

EC Number:

203-812-5

EC Name:

1,3,5-trioxane

Cas Number:

110-88-3

Molecular formula:

C3H6O3

IUPAC Name:

1,3,5-trioxane

Details on test material:

- Name of test material (as cited in study report): C-235
- Physical state: white, crystalline solid
- Analytical purity: approx. 100 %
- Storage condition of test material: under refrigeration

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 Breeding Laboratories
- Age at study initiation: males 41 days, females 58 days
- Mean body weight at test initiation: males 204 g (186-213), females 199 g (187-214)
- Diet (e.g. ad libitum): Purina Rodent Laboratory Chow
- Water (e.g. ad libitum): tap water
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: no data (vapor)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel and glass chambers with a total volume of 1 m3 and an effective volume of 760 liters.
- Air flow rate: The chambers were operated dynamically at an airflow rate of 190 liters per minute. This flow provided one complete air change every 5.3 minutes and a 99% equilibrium time of 24.2 minutes
- Generation of 100 and 1000 ppm trioxane: For generation levels at 100 and 1000 ppm, the material was placed in a 1000 ml three-neck round-bottom flask. The flask used for the low-exposure level was maintained at room temperature, while the flask for the mid-exposure level was kept at 50°C by a temperature regulated water bath. Compressed house-line air was used to sweep the atmosphere enriched with vapour from the flask into the inlet portal of the exposure chamber where it was diluted with room-supplied air.
- Generation of 5000 ppm trioxane: For generation of an exposure level at 5000 ppm test substance, the material was placed in a 4000 ml flask. This flask was also maintained at 50°C using a water bath. Room air was drawn through the bulk of the solid test material, and the resultant airstream with vapour was passed undiluted into the inlet portal of the exposure chamber.
- Determination of the nominal exposure concentration: The generation apparatus and test material were weighed before and after each exposure. The difference in weight represented the total amount of test material delivered into the chamber. This divided by the total volume of air, yielded the nominal exposure concentration.


TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography
- Samples taken from breathing zone: yes, samples were collected for four times daily at a flow rate of 1 liter/minute for 10, 5 and 2.5 minutes for the 100, 1000 and 5000 ppm chambers, respectively. Duplicate samples, taken to clarify values significantly (more than + 20%) at variance with the target concentration, were drawn when necessary. The presence or absence of particulate was checked once each week using a Royco Portable Particle Monitor.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The treated groups were exposed to a cumulative mean analytical concentration of 103, 984 and 4940 ppm of test substance, which were in very good agreement with the respective target concentrations of 100, 1000 and 5000 ppm.

Duration of treatment / exposure:

6 hrs/day, five days/week, for 2 weeks

Frequency of treatment:

The animals were subjected to a total of 10 exposures

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100, 1000 and 5000 ppm
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
103, 984, 4940 ppm
Basis:
analytical conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0.38, 3.62, 18.18 mg/l
Basis:
other: Conversion of analytical concentrations into mg/l according to the "Toxicologist´s Pocket Handbook" (Derelanko MJ, CRC Press, 2000, page 60), and considering a molecular weight for trioxan of 90.08 g/mol.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were examined twice a day for mortalities and clinical symptoms of toxicity; detailed physical examinations were conducted twice a week.


BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded prior test initiation, and thereafter, prior and after the first, the fifth and the tenth exposure.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the termination of the exposure period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: hemoglobin, hematocrit, erythrocytes, platelets, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, clotting time, total and differential leukocytes, erythrocyte morphology, bone marrow differential (control and high concentration level only).


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- How many animals: all
- Parameters checked: serum glutamic pyruvic transaminase (GPT), blood urea nitrogen, fasting glucose, total protein, albumin, globulin, albumin/globulin ratio.

Sacrifice and pathology:

GROSS PATHOLOGY:
At study termination, all animals were sacrificed for the purpose of necropsy and were subjected to gross pathology. The external surface, all orifices, the cranial cavity, the carcass, the external surfaces of brain and spinal cord, the thoracic, abdominal and pelvic cavities, the viscera and the cervical tissues were examined.

ORGAN WEIGHTS:
Selected organs (brain, heart, kidneys, liver, ovaries, testes and epididymes, spleen) were weighed and organ/body weight ratios were calculated.

HISTOPATHOLOGY:
From a series of tissues collected and preserved in 10% neutral buffered formalin (except for bone marrow which was preserved in absolute methanol), following tissues were selected from the control and high exposure level animals for further histopathological examination: right kidney, liver, lungs, peribronchial lymph nodes, nasal turbinates, thymus and bone marrow smear. For examination, the tissues samples were sectioned and stained with Hematoxylin and Eosin except for the bone marrow smears, which were stained with Wright´s stain.

Statistics:

Body weight data, hematology and clinical chemistry parameters, organ weights and organ/body weight ratios were analyzed. Mean values of the treated groups were compared to control at each time interval. Statistically significant differences from control were noticed.
Statistical evaluation of equality of means was made by the appropriate one way analysis of variance technique, followed by a multiple comparison procedure if needed. Bartlett's test was performed to determine if groups had equal variance. If the variances were equal, parametric procedures were used; if not, nonparametric procedures were used. The parametric procedures were the standard one way ANOVA using the F distribution to assess significance. If significant differences among the means were indicated, Dunnett's test was used to determine which means were significantly different from the control. If a non parametric procedure for testing equality of means was needed, the Kruskal-Wallis test was used, and if differences were indicated a summed rank test (Dunn) was used to determine which treatments differed from the control.
A statistical test for trend in the dose levels was also performed. In the parametric case ( i .e . equal variance) standard regression techniques with a tes for trend and lack of fit were used. In the non parametric case Jonckheere's test for monotonic trend was used.
The test for equal variance (Bartlett's) was conducted at the 1%, two-sided risk level. All other statistical tests were conducted at the 5% and 1%, two-sided risk level.

Results and discussion

Results of examinations

Details on results:

MORTALITIES AND CLINICAL SYMPTOMS OF TOXICITY:
- All animals survived.
- Treatment-related symptoms seen in all treated groups (both sexes) consisted of increased secretory responses (lacrimation and mucoid nasal discharge). In the high exposure group further symptoms included reduced righting reflex and grip strength as well as persistent pupillary constriction. Approximately 50% of all high-exposure animals exhibited respiratory impairment on day 12.

BODY WEIGHT (see table 1):
- A treatment-related decrease in mean body weights throughout most study period was reported for males and females of the high exposure group. The differences from control ranged from approximately -4% on the day following the first exposure (females) to ca. -13% on the day prior to the last exposure (both sexes).
- In addition, slight reductions in body weights were also noted in the low exposure group (females only) and the mid exposure group (both sexes) on the day prior to the last exposure.

HEMATOLOGICAL PARAMETERS (see table 2):
- Treatment-related changes in hematological parameters were reported for both, males and females of the high exposure group.
- Hemoglobin, hematocrit and erythrocyte counts were statistically significantly increased for both males (+12, +13 and +18%, respectively) and females (+9, +10 and +11%, respectively) compared to control.
- The mean total leukocyte counts were reduced for both males (-55%) and females (-23%) in the high exposure group, with the reduction being statistically significant for the males only.
- The differencial leukocyte count for the high exposure group revealed that the lymphocyte counts were elevated whereas the segmented neutrophils were decreased, relative to control values.

CLINICAL-CHEMICAL PARAMETERS (see table 3):
- Treatment-related changes in clinical chemical parameters were reported for both, males and females of the high exposure group.
- Slight to statistically significant increases in mean serum glutamic pyruvic transaminase, total protein and albumin values, and a decrease in fasting glucose values were reported for these animals when compared to their respective controls.

GROSS PATHOLOGY:
- Gross pathological examination was inconspicuous and revealed no treatment-related changes in the test groups.

ORGAN WEIGHTS:
- The mean terminal body weights of the treated animals were slightly to statistically significantly decreased when compared to controls, in a treatment-related pattern; the differences from control were about -14% for the high exposure males and females.
- Mean absolute and relative spleen weights were decreased for the animals of all treated groups; both sexes were affected in the mid and high exposure groups whereas in the low exposure group, only the males were affected. No histopathological examination of this organ was done.
- Increased mean relative weights for all other weighed organs (brain, heart, kidneys, liver, ovaries, testes and epididymes) were associated with the significantly decreased mean terminal body weights exhibited by the animals. In the animals of both sexes of the low and mid exposure levels, only a decrease in mean absolute and relative spleen weights was observed; all other findings occurred sporadically and were not considered to be related to exposure.

HISTOPATHOLOGY:
- Histopathology revealed that the mucosa of the anterior nasal cavity from the high exposure males and females showed squamous metaplasia with necrosis and desquamation (see table 4). Acute rhinitis with neutrophile exudation into the nasal cavity was a concomitant change.
- Other changes revealed by histopathology were sporadically seen in both, control and high exposure animals and were not treatment-related.
- Bone marrow differential counts revealed no differences among groups which were considered to be treatment-related. No abnormal cells were seen, and all expected normal cell types were present. There were no differences in cellular degeneration, necrosis, or cell type distributions; variations in distribution were within acceptable limits.

Effect levels

1

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

3.62 mg/L air (analytical)

Sex:

male/female

Basis for effect level:

other: Effects on hematopoietic system and variations on clinical, biochemical parameters in the highest dose group

2

Dose descriptor:

LOAEC

Remarks:

local

Effect level:

< 0.38 mg/L air

Sex:

male/female

Basis for effect level:

other: based on respiratory tract irritation at all tested dose levels.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1. Body weight data

Weights (g)
Males	Prior test start	Prior to 1^st exposure	After 1^st exposure	Prior exposure 5	After exposure 5	Prior exposure 12
0 ppm	162	202	189	224	214	276
103 ppm	163	203	190	226	215	278
984 ppm	162	202	187	223	212	268
4940 ppm	163	207	189	202**	193**	239**
Females
0 ppm	183	201	191	198	193	228
103 ppm	182	197	184	205	195	219
984 ppm	183	199	185	203	192	213
4940 ppm	183	199	183	189	179	200**

** = p < 0.01

Table 2. Hematological parameters

Dose (ppm)	HGB (g/dl)	HCT (%)	RBC (106/mm)	Platelets (105/mm)	MCV (µ3/cell)	MCH (pg/cell)	MCHC (g/dl)	Clot T (min.)	WBC (103/mm)
Males
0	13.6±0.3	40±1	6.20±0.14	15.22±3.75	65±1	22	33.9±0.2	1.4±0.5	13.9±2.4
103	14.0±0.4	41±1	6.28±0.31	13.43±0.89	66±1	22.3	33.9±0.3	1.6±0.2	13.8±2.1
984	13.9±0.5	41±1	6.11±0.27	14.16±3.73	67±2	22.7	34.1±0.4	1.4±0.2	13.3±3.4
4940	15.2±0.8**	45±3**	6.93±0.42**	15.14±2.64	64±1	22	34.2±0.3	1.7±0.3	6.2±1.2**
Females
0	14.1±0.7	42±2	6.45±0.38	10.77±1.62	65±2	21.9	33.7±0.6	1.6±0.2	11.0±1.6
103	14.5±0.3	43±1	6.76±0.19	13.24±1.29	64±3	21.5	33.3±0.5	1.5±0.0	11.0±1.0
984	14.8±0.3	44±1	6.79±0.33	13.26±2.91	65±2	21.8	33.8±0.4	1.7±0.3	10.8±3.8
4940	15.40.6**	46±1**	7.13±0.17**	11.46±2.59	64±1	21.7	33.7±0.5	1.5±0.4	8.5±3.1

** p<0.01

Table 3. Clinical chemistry parameters

Dose (ppm)	SGPT (IU/L)	BUN (mg/dl)	Glucose (mg/dl)	Total protein (g/dl)	Albumin/ globulin
Males
0	29±5	22.8±1.3	149±16	5.4±0.1	1.7±0.2
103	32±3	21.9±2.9	148±8	5.7±0.2	1.6±0.1
984	42±16	22.7±2.3	151±6	5.5±0.3	1.7±0.3
4940	44±6	24.0±1.7	132±9	5.7±0.1	1.7±0.3
Females
0	26±3	24.7±2.5	170±16	5.5±0.2	1.6±0.2
103	26±3	23.0±2.6	153±9	5.8±0.2	1.5±0.1
984	34±10	24.5±1.9	165±20	5.8±0.3*	1.5±0.2
4940	38±11	24.0±1.4	135±13*	5.9±0.2	1.6±0.1

* p<0.05

Table 4. Upper respiratory tract findings:

Findings in the nasal turbinates		Concentration levels
		0 ppm	103 ppm	984 ppm	4940 ppm
Acute rhinitis		0/10	3/10	6/10	7/10
Mucosa	Squamous metaplasia	0/10	0/10	8/10	10/10
	Erosion	0/10	0/10	7/10	10/10
	Hyperplasia	0/10	0/10	2/10	0/10