Di(morpholin-4-yl) disulphide

Endpoint summary

Description of key information

In a 90-day inhalation study (OECD 412), the NOAEC (No-Observed-Adverse-Effect-Concentration) for local effects was considered to be 0.507 mg 4,4’-Dithiodimorpholine/m3 air based on the effects observed in the larynx. The NOAEC for systemic effects was considered to be 8.38 mg 4,4’-Dithiodimorpholine/m3 air. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14-May-2013 to 31-Jan-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended rodent test system.
- Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals per Group: 20 males and 20 females in each of groups 1 and 4, 15 males and 15 females in each of groups 2 and 3
- Age at Delivery: 8 weeks
- Body Weight Range at Delivery: The weight variation in animals did not exceed ±10% of the mean weight of the corresponding sex.
Identification: Acclimatization: Cage card and tail mark (later ear tattoo) during acclimatization and cage card and individual ear tattoo during treatment.
- Randomization: Computer-generated random algorithm.
- Acclimatization: At least five days under test conditions after health examination. Only animals without any visible signs of illness were used for the study. Animals were accustomed to the restraining tubes for 3 daily periods of approximately 1, 2, and 4 hours, respectively.

ENVIRONMENTAL CONDITIONS

- Conditions: Optimal hygienic conditions behind a barrier system. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred, usually following room cleaning, which was considered not to have any influence on the study. These data were not reported but were retained in the raw data. The light cycle was set to 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.
- Accommodation: In groups of maximally four in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding ("Lignocel" J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK).
- Diet: Pelleted standard Harlan Teklad 2914C (batch nos. 52/12 and 29/13) rodent maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum except during the periods when the animals were restrained in the exposure tubes and prior to blood sampling for clinical laboratory investigations. Results of respective analyses for contaminants are archived at Harlan Laboratories Ltd. - Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles except during the periods when the animals were restrained in the exposure tubes. Results of bacteriological assay, chemical and contaminant analyses of respective samples are archived at Harlan Laboratories Ltd.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: - Determination of Particle Size Distribution
The particle size distribution was determined gravimetrically weekly in each of groups 3 and 4. In addition, impactor samples were collected over several days of exposures in group 3. The determination of the particle size distribution was not feasible in group 2 due to the low aerosol concentration.
The cumulative particle size distribution of the test aerosol was determined using a cascade impactor. The test aerosol was impacted at each stage onto an appropriate medium and the particle size distribution of the test item in the generated aerosol was measured by gravimetrically analyzing the test item deposited on each stage of the cascade impactor. The airflow rate through the impactor was 1 L/min.
The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) were calculated on the basis of the gravimetrical results from the impactor, using Microsoft Excel® software (Microsoft Corporation / USA). The target ranges were 1 to 3 µm for the MMAD and 1.5 to 3 for the GSD.

Details on inhalation exposure:

METHOD

- Method: Inhalation by nose-only, flow-past exposure.
- Rationale for Method: Inhalation is a possible route for human exposure.

TARGET AEROSOL CONCENTRATIONS

Group 1: 0 mg/m3 air
Group 2: 0.10 mg/m3 air
Group 3: 0.50 mg/m3 air
Group 4: 8.0 mg/m3 air

Animals of group 1 were treated with compressed, filtered, dried air under the same conditions as animals exposed to the test item.

Target concentrations were selected by the Sponsor and Harlan Laboratories based on the results of a 2-week inhalation study performed at Harlan Laboratories (study D68135).

INHALATION EXPOSURE SYSTEM

Inhalation exposure was performed using a flow-past system. Ports for animal exposure were positioned radially around the nose-only, flow-past exposure chamber on several different levels. The animals were confined separately in restraining tubes. The aerosol was discharged con¬stantly through the exposure system and exhausted using a tubing/filter system.

The exposure system ensured a uniform distribution and provided a constant flow of test material to each exposure tube. The flow of air at each tube was 1.0 L/min, which was sufficient to minimize re-breathing of the test aerosol as it was more than twice the respiratory minute volume of a rat.

Before commencement of the exposure of the animals, technical trials were conducted (without animals) using the inhalation system foreseen for the study. The technical trials were conducted in a GLP certified laboratory, but partly carried out before the study initiation date. Therefore this part is excluded from the statement of GLP compliance. Technical trial data were docu¬mented in the raw data after review but were not reported.

TEST AEROSOL GENERATION

A dust aerosol was generated from the test item with compressed, filtered and dried air using a CR3020 rotating brush aerosol generator connected to a micronizing jet mill. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutralizer. The aerosol concentrations of the test item for groups 3 and 2 were achieved by serial dilution with compressed, filtered, dried air of a higher aerosol concentration using an air vacuum device. The generated test aerosol was diluted as necessary with compressed, filtered and dried air to achieve the concentrations required for this study. Technical details were documented in the raw data.

EXPOSURE SYSTEM MONITORING

Aerosol concentration, particle size distribution, relative humidity, temperature and oxygen con¬centration were measured on test aerosol samples taken at a representative exposure port.

All airflow rates (including those for concentration and particle size measurements) were deter¬mined using calibrated gas meters and pressure gauges or flow meters.

- Determination of Nominal Aerosol Concentration
The test item usage was measured during each exposure in groups 2 to 4 by weighing the generator cylinders containing the test item before and after exposure to determine the quantity of test item used. The weight used was then divided by the total air-flow volume to give the nominal concentration under consideration of the dilution with the air vacuum device. These data were used for the purpose of monitoring the performance of the generation system.

- Gravimetric Determination of Aerosol Concentration
Gravimetric determination of the aerosol concentration was performed twice in groups 2 and 3 (sampling in parallel) and three times during each exposure for group 4. Additional samples were collected if considered necessary.
Test aerosol samples were collected onto a suitable filter using a stainless steel filter sampling device if feasible. Sampling flow was similar to the air flow rate per exposure port. The duration of sampling was sufficient to ensure reliable results. The filters were weighed before and imme¬diately after sampling using a calibrated balance. The gravimetric aerosol concentration was calculated from the amount of test item present on the filter and the sample volume.

- Oxygen Concentration
The oxygen concentration in the chamber was measured continuously during each exposure using a calibrated device. Additionally, values were recorded in general hourly during each exposure. The oxygen concentration was maintained above 19% during the exposure period.

- Relative Humidity / Temperature
The relative humidity and temperature in the chamber were measured continuously during each exposure using a calibrated device. Additionally, values were recorded in general hourly during each exposure.

- Airflow Rate
The exposure airflow rate was adjusted as appropriate before the start of the exposure using calibrated flow-meters and/or pressure gauges. The actual airflow rate was monitored hourly during each exposure. Additional measurements were performed if considered necessary.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analyses of test aerosol samples were performed in groups 2 to 4 using one filter sample per group per week from gravimetric determinations. Additional samples were collected in the first half of treatment period for group 2 but also in group 3.
The samples were transferred into labeled appropriate vials, forwarded at ambient temperature to the scientist responsible for formulation analysis (Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen / Switzerland) and were directly analyzed or stored in the refrigerator (5±3°C) until analysis. They were analyzed for the test item using a LC-MS method supplied by the Sponsor and adapted by Harlan Laboratories Ltd.

The test item was used as analytical standard.

Duration of treatment / exposure:

6 hours per day

Frequency of treatment:

5 days per week at approximately 24-hour intervals for 13 consecutive weeks.

Remarks:

Doses / Concentrations:
0, 0.10, 0.50 and 8.0 mg/m3 air
Basis:
other: Target aerosol concentration

No. of animals per sex per dose:

20 males and 20 females in each of groups 1 and 4 (0 and 8.0 mg/m3 air)
15 males and 15 females in each of groups 2 and 3 (0.10 and 0.50 mg/m3 air)

Details on study design:

The purpose of this inhalation study was to assess the cumulative toxicity of 4,4’-Dithiodimorpholine when administered 6 hours daily (5 days per week) to rats by nose-only, flow-past inhalation exposure for a period of 13 weeks. The reversibility or progression of any test item related effects or any delayed toxicity was assessed during a 6-week exposure free recovery period. This study was designed to indicate potential target organs and to provide a rational basis for the assessment of the toxicological risk to man.

Groups of 10 male and 10 female Wistar rats each were exposed by nose-only flow-past inhalation to target concentrations of 0.10, 0.50 and 8.0 mg 4,4’-Dithiodimorpholine/m3 in each of groups 2 to 4, respectively. The rats of the control group were exposed to filtered air only (group 1). A additional rats of each sex in all groups were kept for a 6-week recovery period.

Mortality, clinical signs, food consumption, body weights, ophthalmoscopic findings, clinical laboratory investigations, organ weights, macroscopic and microscopic findings were recorded.

Positive control:

Not required

Observations and examinations performed and frequency:

The observations listed below were recorded.

- Viability / Mortality: Twice daily during treatment (including week-ends), once daily during acclimatization and recovery.
- Clinical Signs: Recorded twice daily before and after exposure, once daily on week-ends, and once weekly during acclimatization and recovery.
- Food Consumption: Recorded weekly (per cage) during acclimatization, treatment and recovery.
- Body Weights: Recorded twice weekly (each individual animal) and weekly during acclimatization and recovery.
- Ophthalmoscopy: Performed using a direct ophthalmoscope during acclimatization (17-Mai-2013 - all animals) and in week 13 of treatment (16-Aug-2013 - allocation A animals)

Sacrifice and pathology:

NECROPSY

Sacrifice:

- After 13 Weeks of Treatment:
20-Aug-2013 (allocation A males)
21-Aug-2013 (allocation A females)
- After 6 Weeks of Recovery:
01-Oct-2013 (allocation B males)
02-Oct-2013 (allocation B females)

All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals surviving to the end of the observation period were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination.

Samples of the following tissues and organs were collected from all animals at necropsy and, unless otherwise indicated, fixed in neutral phosphate buffered 4% formaldehyde solution. Additional tissues were retained in accordance with standard operating procedures but were not processed or examined further.

- Adrenal glands
- Aorta
- Bone (sternum, femur including joint)
- Bone marrow (femur)
- Brain - including section of medulla/pons, cerebral and cerebellar cortex
- Cecum
- Colon
- Duodenum
- Epididymides (fixed in modified Davidson's solution)
- Esophagus
- Eyes w/optic nerve (fixed in Davidson's solution)
- Harderian gland (fixed in Davidson's solution)
- Heart including auricles
- Ileum, with Peyer's patches
- Jejunum with Peyer's patches
- Kidneys
- Larynx
- Lacrimal gland, exorbital
- Liver
- Lungs, all lobes including main bronchi, filled w/formalin at necropsy
- Lymph nodes - bronchial, mandibular
- Mammary gland area
- Nasal cavity Histopathological examination of the nasal cavities includes examination of the nasal-associated lymphoid tissue (NALT).
- Ovaries
- Pancreas
- Pharynx including nasopharyngeal duct
- Pituitary gland
- Prostate gland
- Rectum
- Salivary glands - mandibular, sublingual
- Sciatic nerve
- Seminal vesicles incl. coagulating glands
- Skeletal muscle
- Skin
- Spinal cord - cervical, midthoracic, lumbar
- Spleen
- Stomach
- Testes (fixed in modified Davidson's solution)
- Thymus
- Thyroid (incl. parathyroid gland, if possible)
- Tongue
- Trachea - adjacent to larynx and carina and bifurcation
- Ureter
- Urinary bladder, filled w/formalin at necropsy
- Uterus (incl. oviducts, cervix and vagina)
- All gross lesions

ORGAN WEIGHTS

The weights of the organs from all animals listed below were recorded on the scheduled dates of necropsy listed and their organ to terminal body weight ratios as well as organ to brain weight ratios determined.

- Adrenal glands
- Brain - including section of medulla/pons, cerebral and cerebellar cortex
- Heart including auricles
- Kidneys
- Liver
- Lungs, all lobes including main bronchi, filled w/formalin at necropsy
- Spleen
- Testes (fixed in modified Davidson's solution)
- Thymus
- Uterus (incl. oviducts, cervix and vagina)

HISTOTECHNIQUE

All organ and tissue samples listed above in the section “Necropsy” to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. The same applied to the larynx, lungs, and pharynx of all animals of allocation A of groups 2 and 3 and the nasal cavity of all animals of groups 2 and 3 was processed and embedded.

All slides were sent to the responsible for histopathology at Peter Millar Associates Ltd. After completion of the study all wax blocks and remaining wet tissues were returned to the test facility (along with the raw data).

HISTOPATHOLOGY

Slides of all organs and tissues listed above collected at terminal sacrifice from the animals of the control and high-dose groups (allocation A) were examined by the study pathologist. The same applied to all occurring gross lesions (allocation A and B). A peer review of findings was performed.

As test item-related morphologic changes were detected in the larynx of the high-dose animals, this organ from the mid- and low-dose group (Allocation A) and from all recovery animals (Allocation B) was examined to establish a no-observed-effect level and/or a no-observed-adverse-effect level.

Other examinations:

CLINICAL LABORATORY INVESTIGATIONS

Blood and Urine Sampling:

- After 13 Weeks of Treatment:
20-Aug-2013 (allocation A males)
21-Aug-2013 (allocation A females)

- After 6 Weeks of Recovery:
01-Oct-2013 (allocation B males)
02-Oct-2013 (allocation B females)

Blood samples were drawn sublingually from all allocated animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

The following hematology parameters were determined:

Complete Blood Cell Count:
- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Red cell volume distribution width
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Hemoglobin concentration distribution width
- Reticulocyte count
- Reticulocyte maturity index (low, medium, high fluorescence)
- Leukocyte count, total
- Differential leukocyte count:
- Neutrophils
- Eosinophils
- Basophils
- Lymphocytes
- Monocytes
- Large unstained cells
- Platelet count

Coagulation
- Prothrombin time (= Thromboplastin time)
- Activated partial Thromboplastin time

The following clinical biochemistry parameters were determined:

- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Phospholipids
- Aspartate aminotransferase
- Alanine aminotransferase
- Lactate dehydrogenase
- Alkaline phosphatase
- Gamma-glutamyl-transferase
- Creatine kinase
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio

The following urine parameters were determined:

Physical Examination
- Urine volume (18 hour)
- Specific gravity (relative density)
- Color
- Appearance

Chemical Examination
- pH value
- Nitrite
- Protein
- Glucose
- Ketones
- Urobilinogen
- Bilirubin
- Erythrocytes
- Leukocytes

Statistics:

The following statistical methods were used to analyze the food consumption, body weight, ophthalmoscopic examinations, macroscopic findings, organ weights and ratios, as well as clinical laboratory data:

- 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 to the ophthalmoscopic and macroscopic findings.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A decreased body weight gain was noted in both sexes of groups 3 and 4 during the treatment period, in males limited to the first two weeks of treatment

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased kidney weight in males of group 4 was considered to be of no toxicological relevance in the absence of corresponding histopathological alterations.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Minor findings in the larynx in groups 2 and 3 considered non-adverse. Erosion and ulceration in both sexes in group 4 along with moderate to marked severity for the squamous metaplasia in the larynx considered adverse.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

1. INHALATION TECHNICAL DATA

Numeric results are presented in the following format, where appropriate:
Mean value ± standard deviation (number of determinations [n], coefficient of variation [CV])

EXPOSURE CONDITIONS

Temperature, relative humidity and oxygen concentration levels were satisfactory for this type of study and were similar across all groups. The low relative humidity was a consequence of the usage of dried air for aerosol generation. Dried air was used to reduce agglomeration of particles and to optimize the particle size distribution.

Tabulated results are presented in the section “Any other information on results incl. tables” below:

TEST ATMOSPHERE CONCENTRATIONS

The mean nominal aerosol concentrations were 0.464, 1.91 and 39.3 mg 4,4’-Dithiodimorpholine powder/m3 air in groups 2, 3 and 4, respectively.

The aerosol concentrations of 4,4’-Dithiodimorpholine were close to the respective target concentrations for all groups. The concentrations were quite stable during the whole exposure period in group 4 as demonstrated by the low CV (13%). Higher variations for the gravimetrically determined mean aerosol concentrations towards low dose group were considered to result from the lower absolute amount of the test item on the filters (approximately 35 µg in group 2 and approximately 180 µg in group 3).

The chemically determined mean aerosol concentrations confirmed the gravimetric determinations. The values on the corresponding days of analysis were similar. The relative difference for the mean values was less than 10%.

Tabulated results are presented in the section “Any other information on results incl. tables” below:

PARTICLE SIZE DISTRIBUTION

Mean values for the mass median aerodynamic diameters (MMADs) of slightly below 2 µm were noted in groups 3 and 4 as determined by a cascade impactor and also the mean Geometric Standard Deviations (GSDs) were within the target range of 1.5 to 3. Therefore, deposition of the particles can be assumed to have occurred in the upper and lower respiratory tract as all MMADs were well within the target range. Single determinations of the MMAD and the GSD were above the respective target range but considered to be negligible as more than 67% of the particles were considered to be below 3 µm.

The aerosol concentration in group 2 was too low for this determination but as the mean MMAD and the mean GSD in groups 3 and 4 were similar and because the aerosol for group 2 was generated from the same aerosol as for those groups it can be concluded that the particle size distribution was also similar in that group.

In conclusion, the particle size distributions obtained were considered to be suitable for inhalation toxicity testing.

Tabulated results are presented in the section “Any other information on results incl. tables” below:

2. OBSERVATIONS

VIABILITY/MORTALITY

There were no unscheduled deaths during the course of the study.

CLINICAL SIGNS

There were no clinical signs that were considered to be related to treatment.

The observation of increased activity for 2 females (animal nos. 124 and 135 of group 4) during 9 days at the end of the treatment period, as well as hair loss observed in two females (animal no. 94 of group 2 and animal no. 140 of group 4) from week 8 to the end of treatment was considered fortuitous.

FOOD CONSUMPTION

The food intake during the treatment period was similar across all groups.

Slightly increased food consumption was noted in group 4 males during the recovery period attaining statistical significance on one occasion. This finding is considered incidental, as the body weight of the recovery satellite of group 4 was generally higher than that of the concurrent control group, and in consequence also the food consumption of these animals.

BODY WEIGHTS

There were no relevant treatment-related effects on absolute body weight during the course of this study. Statistical significant differences to controls for males in group 3 were considered to be fortuitous and related to a low body weight at treatment start (-2.6%).

However, a treatment-related decreased body weight gain was noted in both sexes of groups 3 and 4. A reduced body weight gain in males was recorded during the first 2 weeks attaining generally statistical significance. Differences to controls diminished during the course of the treatment period resulting in a similar body weight gain at the end of the treatment period. In females, a lower body weight gain was observed during the course of the treatment period attaining statistical significance on various occasions.

Increased body weight gain was noted in males and females of group 4 during the recovery period attaining statistical significance on some occasions for group 4.

OPHTHALMOSCOPIC EXAMINATION

Some ophthalmoscopic findings were observed but were not considered to be attributable to exposure with the test item.

3. CLINICAL LABORATORY INVESTIGATIONS

HEMATOLOGY

There were no changes in hematology parameters that were considered to be related to treatment.

Some inter-group variations occasionally achieved statistical significance but they were only seen in one sex or were within historical control data and did not show a relationship to the concentration.


CLINICAL BIOCHEMISTRY

There were no changes in clinical biochemistry parameters that were considered to be related to treatment.

Some inter-group variations occasionally achieved statistical significance but they were only seen in one sex or were within historical control data and did not show a relationship to the concentration.

URINALYSIS

There were no changes in urinalysis parameters that were considered to be related to treatment.

4. PATHOLOGY

ORGAN WEIGHTS

Relative kidney weight was slightly but statistically significantly increased in females of group 3 (+6%) and in males of group 4 (11%) when compared with controls. In the absence of any histopathologic findings in the kidneys, this finding is considered of no toxicological relevance.

The only other statistically significant difference in organ weight parameters was considered fortuitous and unrelated to exposure, as it did not occur in a concentration-related manner.

MACROSCOPIC FINDINGS

There were no treatment-related macroscopic findings.

MICROSCOPIC FINDINGS

Minimal to marked focal squamous metaplasia with associated minimal or mild submucosal inflammation were present in the larynx of animals from all the test item-treated groups. The incidence and severity of the findings were distributed in a concentration-related manner. These findings were also accompanied by minimal or mild focal erosion or ulceration in a male and 2 females of group 4 and minimal or mild focal epithelial hyperplasia of the squamous epithelium in a male of group 2 and two males of group 3 and all the males and nine females of group 4.

After a six-week period of recovery, minimal to moderate focal squamous metaplasia, minimal focal hyperplasia of the squamous epithelium and minimal submucosal inflammation were still present in animals of both sexes of group 4 but were not observed in animals of groups 2 and 3. Indeed, treatment-related changes were still present in the larynx but were confined to animals of group 4. The findings were generally present with a lower severity grade than in animals at the end of treatment. This was considered to indicate that, given sufficient time, the findings would resolve completely.

There were no other treatment-related findings. All of the other histopathological findings were considered to have arisen spontaneously or post mortem.

Dose descriptor:

NOAEC

Remarks:

for local effects

Effect level:

0.507 mg/m³ air

Sex:

male/female

Basis for effect level:

other: .

Dose descriptor:

NOAEC

Remarks:

for systemic effects

Effect level:

8.38 mg/m³ air

Sex:

male/female

Basis for effect level:

other: No adverse systemic effect was observed at the highest dose.

Critical effects observed:

not specified

1. INHALATION TECHNICAL DATA

 

Numeric results are presented in the following format, where appropriate:

Mean value ± standard deviation (number of determinations [n], coefficient of variation [CV])

 

EXPOSURE CONDITIONS

 

Group	Temperature [°C]	Relative Humidity [%]	Oxygen Concentration [%]
1	23.2 ± 0.1 (n=394)	1.9 ± 0.1 (n=394)	20.1 ± 0.0 (n=394)
2	23.4 ± 0.1 (n=394)	1.8 ± 0.3 (n=394)	20.1 ± 0.0 (n=394)
3	22.9 ± 0.1 (n=394)	2.3 ± 0.2 (n=394)	20.5 ± 0.1 (n=394)
4	22.5 ± 0.1 (n=394)	1.0 ± 0.3 (n=394)	20.6 ± 0.0 (n=394)

 

TEST ATMOSPHERE CONCENTRATIONS

 

Group	Chemically Determined Mean Aerosol Concentration [mg/m^3air]	Gravimetrically Determined Mean Aerosol Concentration [mg/m^3air]	Target Aerosol Concentration [mg/m^3air]	Gravimetry Relative to Target
2	0.108 ± 0.025 (n=20, CV=23.3%)	0.099 ± 0.035 (n=131, CV=35.5%)	0.10	99.1%
3	0.485 ± 0.109 (n=13, CV=22.5%)	0.507 ± 0.112 (n=132, CV=22.0%)	0.50	101.6%
4	8.89 ± 1.14 (n=13, CV=12.9%)	8.38 ± 1.10 (n=198, CV=13.1%)	8.0	104.7%

 

 

PARTICLE SIZE DISTRIBUTION

 

Group	Mean (Range of) MMAD [µm]	Mean (Range of) GSD	Number of Determinations	Mean Percentage of Particles <3 µm
3	1.86 (1.36 - 3.17)	2.92 (1.96 - 3.68)	13	67.3%
4	1.89 (1.63 - 2.33)	2.36 (2.11 - 2.98)	14	70.4%

 

Conclusions:

Wistar rats were exposed by nose-only flow past inhalation for 6 hours/day, 5 days/week over a period of 13 weeks. Control animals were exposed to air only. Exposure to gravimetrically determined atmosphere concentrations of 0.099, 0.507 and 8.38 mg 4,4’-Dithiodimorpholine/m3 air (confirmed by chemical analysis) resulted in histopathological findings in the larynx at all concentrations. In groups 3 and 4 effects on the body weight were also noted; however, increased body weight gain after the end of treatment demonstrated recovery.

The increased kidney weight in males of group 4 was considered to be of no toxicological relevance in the absence of corresponding histopathological alterations.

Minor findings in the larynx of animals of groups 2 and 3 were considered as non-adverse. The observation of erosion and ulceration in animals of both sexes in group 4 along with a higher severity degree of moderate to marked for the squamous metaplasia in the larynx, however, was considered as adverse. However, the fact that the findings had resolved in groups 2 and 3 at the end of the recovery period, was considered to indicate that, given sufficient time, the findings would resolve completely.

Based on the findings in the larynx, the NOAEC (No-Observed-Adverse-Effect-Concentration) for local effects was considered to be 0.507 mg 4,4’-Dithiodimorpholine/m3 air. The NOAEC for systemic effects was considered to be 8.38 mg 4,4’-Dithiodimorpholine/m3 air.

Executive summary:

The purpose of this inhalation study was to assess the cumulative toxicity of 4,4’-Dithiodimorpholine when administered 6 hours daily (5 days per week) to rats by nose-only, flow-past inhalation exposure for a period of 13 weeks. The reversibility or progression of any test item related effects or any delayed toxicity was assessed during a 6-week exposure free recovery period. This study was designed to indicate potential target organs and to provide a rational basis for the assessment of the toxicological risk to man.

 

Groups of 10 male and 10 female Wistar rats each were exposed by nose-only flow-past inhalation to target concentrations of 0.10, 0.50 and 8.0 mg 4,4’-Dithiodimorpholine/m3 in each of groups 2 to 4, respectively. The rats of the control group were exposed to filtered air only (group 1). A additional rats of each sex in all groups were kept for a 6-week recovery period.

 

Mortality, clinical signs, food consumption, body weights, ophthalmoscopic findings, clinical laboratory investigations, organ weights, macroscopic and microscopic findings were recorded.

 

TECHNICAL DATA

 

Exposure to gravimetrically determined atmosphere concentrations of 0.099, 0.507 and 8.38 mg 4,4’-Dithiodimorpholine/m3 air were achieved in groups 2 to 4, respectively, and were close to the respective targets. The chemically determined mean aerosol concentrations confirmed the gravimetric determinations. Temperature, relative humidity and oxygen concentration during exposure were considered to be suitable for this type of study. The mean MMAD (mean mass aerodynamic diameter) and GSD (geometric standard deviation) were 1.86 ¿m (GSD = 2.92) for group 3 and 1.89 µm (GSD = 2.36) for group 4. In conclusion, the test item was considered to be respirable to rats.

 

BIOLOGICAL DATA

 

A decreased body weight gain was noted in both sexes of groups 3 and 4 during the treatment period. In males, it was limited to the first 2 weeks of treatment. Increased body weight gain was noted in males and females of group 4 during the recovery period.

 

Relative kidney weight was slightly but statistically significantly increased in females of group 3 and in males of group 4 when compared with controls. In the absence of any histopathologic findings in the kidneys, this finding is considered of no toxicological relevance.

 

Histopathological changes in the larynx of both sexes were noted in a concentration-related manner. These findings included focal squamous metaplasia, focal hyperplasia of squamous epithelium, submucosal inflammation and focal erosion/ulceration. The latter finding was noted in group 4 only. After the end of the recovery period, complete recovery was observed in groups 2 and 3 and partial recovery in group 4.

 

There were no further findings that were considered to be related to treatment.

 

CONCLUSION

Based on the findings in the larynx, the NOAEC (No-Observed-Adverse-Effect-Concentration) for local effects was considered to be 0.507 mg 4,4’-Dithiodimorpholine/m3 air. The NOAEC for systemic effects was considered to be 8.38 mg 4,4’-Dithiodimorpholine/m3 air.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

8.38 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Sieber's study is considered to be a reliable study with a klimisch score of 1.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation

Remarks:

other: 2-Week Dose-Range Finding Study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Between 05 and 27-Feb-2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study was conducted in accordance with Good Laboratory Practice principles, however no specific study-related Quality Assurance procedures were performed.

Qualifier:

no guideline available

Principles of method if other than guideline:

In this dose range finding study, 4,4’-Dithiodimorpholine was administered to four groups of 5 male and female Wistar rats by nose-only, flow-past inhalation for a period of 6 hours per day at 5 days/week for two weeks to target concentrations of 3, 15, and 75 mg/m3 air of 4,4’-Dithiodimorpholine in groups 2 to 4, respectively. Based on a premature death in group 4 after 2 days of treatment along with effects on breathing and body weight development at this concentration, the target aerosol concentration was reduced to 45 mg/m3 air from day 6. Animals of group 1 were exposed to air only. Throughout the study all animals were observed for viability, body weight, food consumption clinical signs, ophthalmoscopic findings and clinical laboratory investigations. Necropsy and macroscopic examination of all animals was performed and organ weights were recorded. Microscopic examination was performed thereafter.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended rodent test system.
- Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at Delivery: 8 weeks
- Body Weight Range at Delivery: 219.7 to 244.8 g (males) and 151.9 to 170.0 g (females). The weight variation in animals did not exceed ± 6% of the mean weight of the corresponding sex.
- Identification: Cage card and tail mark.
- Randomization: Computer-generated random algorithm.
- Acclimatization: At least five days under test conditions after health examination. Only animals without any visible signs of illness were used for the study. Animals were accustomed to the restraining tubes for 3 daily periods of approximately 1, 2 and 4 hours, respectively.

ENVIRONMENTAL CONDITIONS
- Conditions: Optimal hygienic conditions behind a barrier system. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values below the relative humidity range occasionally occurred, but were considered not to have any influence on the study. These data were not reported but were retained in the raw data. The light cycle was set to 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.
- Accommodation: In groups of maximally five in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding ("Lignocel" J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kai¬seraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK).
- Diet: Pelleted standard Harlan Teklad 2914C (batch no. 39/12) rodent maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland), was available ad libitum except during the periods when the animals were restrained in the exposure tubes and prior to blood sampling for clinical laboratory investigations. The results of respective analyses for contaminants are archived at Harlan Laboratories Ltd.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles except during the periods when the animals were restrained in the exposure tubes. A bacteriological assay, chemical and contaminant analyses of respective samples were performed. Results are archived at Harlan Laboratories Ltd.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: The target ranges were 1 to 3 µm for the MMAD and 1.5 to 3 for the GSD.

The particle size distribution was determined gravimetrically three to four times in each of groups 2 to 4. The cumulative particle size distribution of the test aerosol was determined using a cascade impactor. The test aerosol was impacted at each stage onto an appropriate medium and the particle size distribution of the test item in the generated aerosol was measured by gravimetrically analyzing the test item deposited on each stage of the cascade impactor. The airflow rate through the impactor was 1 L/min.

The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) were calculated on the basis of the gravimetric results from the impactor, using Microsoft Excel® software (Microsoft Corporation, USA). The mean MMADs and the mean GSDs were calculated from the mean percentage values of the different stages.

Details on inhalation exposure:

METHOD
- Method: Inhalation by nose-only, flow-past exposure.
- Rationale for Method: Inhalation is a possible route for human exposure.

TARGET AEROSOL CONCENTRATIONS

Group 1: 0 mg/m3 air
Group 2: 3 mg/m3 air
Group 3: 15 mg/m3 air
Group 4: 75 mg/m3 air (until day 3 included; 15-Feb-2013) and 45 mg/m3 air (from day 6 onwards; 18-Feb-2013. There was no treatment on days 4 and 5 due to the 5 days per week administration regimen.)

INHALATION EXPOSURE SYSTEM

Inhalation exposure was performed using a flow-past system. Ports for animal exposure were positioned radially around the nose-only, flow-past exposure chamber on several different levels. The animals were confined separately in restraining tubes. The aerosol was discharged constantly through the exposure system and exhausted using a tubing/filter system.

The exposure system ensured a uniform distribution and provided a constant flow of test material to each exposure tube. The flow of air at each tube was 1.0 L/min, which was sufficient to minimize re-breathing of the test aerosol as it was more than twice the respiratory minute volume of a rat.

Before commencement of the exposure of the animals, technical trials had been conducted (without animals) using the inhalation system foreseen for the study. Technical trial data were documented in the raw data after review but were not reported.

TEST AEROSOL GENERATION

A dust aerosol was generated for group 4 from the test item with compressed, filtered and dried air using a rotating brush aerosol generator connected to a micronizing jet mill. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutralizer. Alternatively, the aerosol concentrations of the test item for groups 2 and 3 were achieved by serial dilution with compressed, filtered, dried air of a higher aerosol concentration using an air vacuum device. The generated test aerosol was diluted as necessary with compressed, filtered and dried air to achieve the concentrations required for this study. Technical details were documented in the raw data.

EXPOSURE SYSTEM MONITORING

Aerosol concentration, particle size distribution, relative humidity, temperature and oxygen concentration were measured on test aerosol samples taken at a representative exposure port.

All airflow rates (including those for concentration and particle size measurements) were determined using calibrated gas meters and pressure gauges or flow meters.

- Determination of Nominal Aerosol Concentration:
The test item usage was measured during each exposure in groups 2 to 4 by weighing the generator cylinders containing the test item before and after exposure to determine the quantity of test item used. The weight used was then divided by the total air-flow volume to give the nominal concentration. These data were used for the purpose of monitoring the performance of the generation system.

- Gravimetric Determination of Aerosol Concentration:
Gravimetric determination of the aerosol concentration was performed three times during each exposure for groups 2 to 4. Additional samples were collected in group 4 for monitoring purposes.
Test aerosol samples were collected onto a Millipore durapore filters (type HVLP) using a stainless steel filter sampling device if feasible. Sampling flow was similar to the air flow rate per exposure port. The duration of sampling was approximately 2 hours. The filters were weighed before and immediately after sampling using a calibrated balance. The gravimetric aerosol concentration was calculated from the amount of test item present on the filter and the sample volume.

- Oxygen Concentration:The oxygen concentration in the chamber was measured continuously during each exposure using a calibrated device. Additionally, values were recorded hourly during each exposure. The oxygen concentration was maintained above 19% during the exposure period.

- Relative Humidity / Temperature: The relative humidity and temperature in the chamber were measured continuously during each exposure using a calibrated device. Additionally, values were recorded hourly during each exposure.

- Airflow Rate: The exposure airflow rate was adjusted as appropriate before the start of the exposure using calibrated flow-meters and / or pressure gauges. The actual airflow rate was monitored hourly during each exposure.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Filter samples from 20 and 21-Feb-2013 of all groups were used outside the scope of this study for the chemical determination of the aerosol concentration within the technical trials of Harlan Laboratories Study D68135 (13-week inhalation).

Duration of treatment / exposure:

6 hours per day
2 consecutive weeks,

Frequency of treatment:

5 days per week at approximately 24-hour intervals for 2 consecutive weeks

Remarks:

Doses / Concentrations:
0, 3, 15 and 75/45 mg/m3 air
Basis:
other: Target Aerosol Concentration

No. of animals per sex per dose:

5 males and 5 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The purpose of this inhalation study was to assess the cumulative toxicity of 4,4’-Dithiodimorpholine when administered 6 hours daily (5 days per week) to rats by nose-only, flow-past inhalation exposure for a period of at least 2 weeks. The results of this study provided preliminary information about the toxicity to evaluate the range of toxicity. The results are used to select the target concentrations for a subsequent 13-week toxicity study.

In this dose range finding study, 4,4’-Dithiodimorpholine was administered to four groups of 5 male and female Wistar rats by nose-only, flow-past inhalation for a period of 6 hours per day at 5 days/week for two weeks to target concentrations of 3, 15, and 75 mg/m3 air of 4,4’-Dithiodimorpholine in groups 2 to 4, respectively. Based on a premature death in group 4 after 2 days of treatment along with effects on breathing and body weight development at this concentration, the target aerosol concentration was reduced to 45 mg/m3 air from day 6. Animals of group 1 were exposed to air only. Throughout the study all animals were observed for viability, body weight, food consumption clinical signs, ophthalmoscopic findings and clinical laboratory investigations. Necropsy and macroscopic examination of all animals was performed and organ weights were recorded. Microscopic examination was performed thereafter.

Positive control:

Not required

Observations and examinations performed and frequency:

The observations listed below were recorded. Additional observations and examinations were performed upon animals judged to be in extremis, showing signs of bad health or unusual findings.

- Viability / Mortality: Twice daily during treatment (including week-ends) and once daily during acclimatization.
- Clinical Signs: Recorded twice daily before and after exposure, once daily on weekends and once weekly during acclimatization.
- Food Consumption: Recorded twice weekly (per cage) during acclimatization and treatment.
- Body Weights: Recorded twice weekly during acclimatization and treatment. An additional measurement was performed after exposure on day 3 of treatment to monitor the health status of the animals of group 4 because these animals showed signs of bad health. These measurements were excluded from the calculation of the mean because all other measurements were recorded before the daily exposure and, therefore, the values would not have been comparable.
- Ophthalmoscopy: Performed using a direct ophthalmoscope once during acclimatization and once during week 2 of treatment.

Sacrifice and pathology:

NECROPSY

All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals surviving to the end of the observation period were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination.

Sacrifice after 2 Weeks: 27-Feb-2013

Samples of the following tissues and organs were collected from all animals at necropsy and, unless otherwise indicated, fixed in neutral phosphate buffered 4% formaldehyde solution. Additional tissues were retained in accordance with standard operating procedures but were not processed or examined further. All fixed tissues were sent to the responsible for histotechnology.

- Adrenal glands
- Aorta
- Bone (sternum, femur including joint)
- Bone marrow (femur)
- Brain - including section of medulla/pons, cerebral and cerebellar cortex
- Cecum
- Colon
- Duodenum
- Epididymides (fixed in Bouin's solution)
- Esophagus
- Eyes w/optic nerve (fixed in Davidson's solution)
- Harderian gland (fixed in Davidson's solution)
- Heart including auricles
- Ileum, with Peyer's patches
- Jejunum with Peyer's patches
- Kidneys
- Larynx
- Lacrimal gland, exorbital
- Liver
- Lungs, including main bronchi, filled w/formalin at necropsy
- Lymph nodes - bronchial
- Lymph nodes - mandibular
- Mammary gland area
- Nasal cavity Ovaries
- Pancreas
- Pharynx including nasopharyngeal duct
- Pituitary gland
- Prostate gland
- Rectum
- Salivary glands - mandibular, sublingual
- Sciatic nerve
- Seminal vesicles incl. coagulating glands
- Skeletal muscle
- Skin
- Spinal cord - cervical, midthoracic, lumbar
- Spleen
- Stomach
- Testes (fixed in Bouin's solution)
- Thymus
- Thyroid (incl. parathyroid gland, if possible)
- Tongue
- Trachea - adjacent to larynx and carina and bifurcation
- Ureter
- Urinary bladder, filled w/formalin at necropsy
- Uterus (incl. oviducts, cervix and vagina)
- All gross lesions

ORGAN WEIGHTS

The weights of the organs from all animals listed below were recorded on the scheduled dates of necropsy listed and their organ to terminal body weight ratios as well as organ to brain weight ratios determined.

- Brain - including section of medulla/pons, cerebral and cerebellar cortex
- Heart including auricles
- Kidneys
- Liver
- Lungs, including main bronchi, filled w/formalin at necropsy
- Lymph nodes - bronchial
- Ovaries
- Spleen
- Testes (fixed in Bouin's solution)

HISTOTECHNIQUE

All organ and tissue samples to be examined by the study pathologist (see list under “Histopathology” below), embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin.

All slides were sent to responsible for histopathology. After completion of the study all wax blocks and remaining wet tissues were returned to the test facility (along with the raw data).


HISTOPATHOLOGY

Slides of all organs and tissues listed below collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the study pathologist.

- Heart including auricles
- Kidneys
- Larynx Lacrimal gland, exorbital
- Liver
- Lungs, including main bronchi, filled w/formalin at necropsy
- Lymph nodes - bronchial
- Nasal cavity Pharynx including nasopharyngeal duct
- Trachea - adjacent to larynx and carina and bifurcation
- All gross lesions

The same applied to all occurring gross lesions and to all animals, which died spontaneously. A peer review of findings was performed.

In addition, because of treatment-related findings observed in animals from the high-dose group, the larynx, lungs, pharynx and nasal cavities were examined from all animals of the low and intermediate-dose groups to establish a no-effect level, if possible.

A histopathology phase report was provided by the responsible for histopathology.

Other examinations:

CLINICAL LABORATORY INVESTIGATIONS

Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

Blood and urine sampling after 2 weeks: 27-Feb-2013

The following hematology parameters were determined:

Complete Blood Cell Count:
- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Red cell volume distribution width
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Hemoglobin concentration distribution width
- Reticulocyte count
- Reticulocyte maturity index (low, medium, high fluorescence)
- Leukocyte count, total
- Differential leukocyte count:
- Neutrophils
- Eosinophils
- Basophils
- Lymphocytes
- Monocytes
- Large unstained cells
- Platelet count

Coagulation
- Prothrombin time (= Thromboplastin time)
- Activated partial Thromboplastin time

The following clinical biochemistry parameters were determined:

- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Phospholipids
- Aspartate aminotransferase
- Alanine aminotransferase
- Lactate dehydrogenase
- Alkaline phosphatase
- Gamma-glutamyl-transferase
- Creatine kinase
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio

The following urine parameters were determined:

Physical Examination
- Urine volume (18 hour)
- Specific gravity (relative density)
- Color
- Appearance

Chemical Examination
- pH value
- Nitrite
- Protein
- Glucose
- Ketones
- Urobilinogen
- Bilirubin
- Erythrocytes
- Leukocytes

Statistics:

The following statistical methods were used to analyze the body weight, ophthalmoscopic examinations, macroscopic findings, organ weights and ratios, as well as clinical laboratory data:

- 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 to the ophthalmoscopic and macroscopic findings.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mainly effects on breathing, ruffled fur and salivation in the high dose group as well as death of one high dose animal

Mortality:

mortality observed, treatment-related

Description (incidence):

Mainly effects on breathing, ruffled fur and salivation in the high dose group as well as death of one high dose animal

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced in the mid and high dose groups

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

reduced in the mid and high dose groups

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative neutrophil counts statistically significantly increased in high dose females

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A statistically significantly increased urea level recorded in high dose males.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Concentration-related effects in the respiratory tract (mainly erosion, ulceration, squamous metaplasia and/or inflammation)

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

1. INHALATION TECHNICAL DATA

Numeric results are presented in the following format, where appropriate:
Mean value ± standard deviation (number of determinations [n], coefficient of variation [CV])

GRAVIMETRIC AEROSOL CONCENTRATION

The aerosol concentrations were close to the respective targets for all groups. They were also stable during the whole treatment period as demonstrated by the low CVs (below 14.6%).

Data on gravimetric aerosol concentrations are presented in the section “Any other information on results incl. tables” below.

PARTICLE SIZE DISTRIBUTION

The mean mass median aerodynamic diameters (MMADs) ranged between 1.58 and 2.93 µm as targeted. Therefore, deposition of the particles can be assumed to have occurred in the upper but also in the lower respiratory tract. Two MMAD values for group 4 were marginally above the upper limit of the target range of 1 to 3 µm (3.05 µm and 3.18 µm) but were still considered to be acceptable based on significant biological findings in that group (mortality, breathing noises and body weight loss). Most Geometric Standard Deviations (GSD) were within the target range of 1 to 3. Only single values were above the upper limit but were still considered acceptable as the mean percentage of particles was above 50% in each group. In conclusion, the particle size distributions obtained were considered to be appropriate for this type of study.

Data on particle size distribution are presented in the section “Any other information on results incl. tables” below.

EXPOSURE CONDITIONS

Temperature, relative humidity and oxygen concentration levels were satisfactory for this type of study and were similar across all groups.

The group means for temperature, relative humidity and oxygen concentration measured during the exposures are presented in the section “Any other information on results incl. tables” below.

2. OBSERVATIONS

VIABILITY/MORTALITY

One female of group 4 (no. 40) exposed to 82.1 mg/m3 air was found dead on study day 3. Death was attributed to severe inflammation and marked erosion/ulceration of the larynx.

In addition, one female of group 2 (no. 29) exposed to 2.85 mg/m3 air was found dead a few days before scheduled necropsy. No factors contributory to death could be identified on histopathological examination.

All other animals survived the scheduled treatment period.

CLINICAL SIGNS

Four out of 5 males and the female animal of group 4 (82.1 / 47.1 mg/m3 air) that died prematurely showed effects on breathing (labored breathing, breathing noises and/or tachpynea) on various occasions, generally during the first week of treatment. In addition, ruffled fur and salivation was noted in those males on several days, generally during the second week of treatment. Salivation was also noted in two females of that group on single days. One male showed also a decreased activity and a prostrate position on single occasions.

No clinical signs were observed in any other group during the course of the study.

FOOD CONSUMPTION

A concentration-related decrease in food intake was noted in males and females of groups 3 (13.8 mg/m3 air) and 4 (82.1 / 47.1 mg/m3 air). In males the decrease was -12% and -28% and in females the decrease was -9% and -18% in groups 3 and 4, respectively, when compared to controls. The food consumption in group 2 was similar to controls.

BODY WEIGHTS

Slight to marked body weight loss was noted in males and females of group 4 (82.1 / 47.1 mg/m3 air) from day 1 to day 4 of treatment (-11% and -3%, respectively). Thereafter, the animals generally gained weight again. Differences to controls attained statistical significance for the absolute weight and the body weight gain in males during the whole treatment period and on single occasions for females. A concentration-related reduced body weight gain was also noted for group 3 (13.8 mg/m3 air). Differences to controls attained statistical significance during the second week of treatment for females.

Body weight development in group 2 was similar to controls.

OPHTHALMOSCOPIC EXAMINATIONS

Ophthalmoscopic examination resulted in no findings that were considered to be test item-related.

3. CLINICAL LABORATORY INVESTIGATIONS

HEMATOLOGY

Absolute and relative neutrophil counts were increased in females group 4 (82.1 / 47.1 mg/m3 air) the latter achieving statistical significance when compared to controls (0.94 vs 0.46 g/L). A dose-effect relation was observed for this parameter.

Some further intergroup-specific variations in hematology occasionally achieved statistical significance but were considered not to be related to treatment with the test item as they were reflecting the historical control data, and/or did not show a concentration-relationship.

CLINICAL BIOCHEMISTRY

A statistically significantly increased urea level was recorded in males of group 4 compared to controls (7.72 vs 5.80 mmol/L).

Some further intergroup-specific variations in clinical biochemistry occasionally achieved statistical significance but were considered not to be related to treatment with the test item as they were reflecting the historical control data, and/or did not show a concentration-relationship.

URINALYSIS

Urinalysis resulted in no test item-related effects.

4. PATHOLOGY

ORGAN WEIGHTS

There were no effects on absolute or relative organ weights that were considered to be related to treatment.

Some statistically significant alterations on organ weights were observed but did not show a concentration-relationship. In addition, the lower terminal body weight in groups 3 and 4 animals compared to controls was considered to be related to a statistically significant decrease in a few absolute organ weights.

MACROSCOPIC FINDINGS

There were no treatment-related macroscopic findings.

MICROSCOPIC FINDINGS

- Premature Decedents:
One female of group 4 (No. 40) exposed to 82.1 mg/m3 air was found dead on study day 3. Marked erosion/ulceration and severe inflammation of the larynx with the presence of moderate to severe intraluminal inflammatory exudates in the upper respiratory tract were considered to have caused death. In the liver there was moderate centrilobular necrosis; a finding considered to have resulted from poor oxygenation of the blood due to the upper respiratory tract changes.

Animal No. 29, a female of group 2 (2.85 mg/m3 air), was the only other premature decedent. It was found dead after ten days of treatment. No factors contributory to death could be identified in the tissues of this animal.

- Larynx:
Minimal to marked focal squamous metaplasia with associated minimal to severe submucosal inflammation was present in the larynx of animals from all the treated groups. The incidence and severity of the two findings were distributed in a concentration-related manner. These findings were also accompanied by minimal to marked focal erosion or ulceration in 2 males and 2 females of group 3 (13.8 mg/m3 air) and 3 males and all the females of group 4 (82.1 / 47.1 mg/m3 air) and minimal to moderate focal epithelial hyperplasia in 2 females of group 2 and all the animals of groups 3 and 4.

- Lungs:
Minimal or mild foreign-body granulomas were present within the airways of all the males and 2 females of group 4 and in a control female. The finding seen in the control probably resulted from inhalation of food or bedding particles. The granulomas consisted of a small number of macrophages, including multinucleated giant cells containing amorphous foreign material, lying in the lumen at the level of the broncho-alveolar junction. These granulomas appeared to have caused no damage to the adjacent lung tissue.

- Nasal Cavities:
Minimal focal mucosal inflammation was present in 4 out of 5 males and 3 out of 5 females of group 4. This finding occurred in the ventral meatus and was associated with minimal focal degeneration of the squamous epithelium in a male and a female animal and minimal focal hyperplasia of the squamous epithelium in 2 out of 5 males. The findings were mainly confined to the most anterior section of nasal cavity but were also seen in the second most anterior section.

In a male and a female animal of group 3 a mild focus of regenerating epithelium was present at the junction of the respiratory and olfactory epithelium in the dorsal meatus of the nasal cavity (Level II). This finding might be associated with treatment but in the absence of a concentration relationship and its location at the junction of two epithelial types it was thought more likely to be spontaneous in origin.

- Pharynx:
Minimal focal squamous metaplasia was present in 1 male and 1 female animal of group 4. In the affected female it was associated with mild submucosal inflammatory cell infiltration and mild focal ulceration.

There were no other treatment-related findings. All of the other histopathological findings were considered to have arisen spontaneously or post mortem.

Dose descriptor:

NOEL

Effect level:

< 3 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the histopathological results, a no-observed-effect-level (NOEL) could not be established

Critical effects observed:

not specified

GRAVIMETRIC AEROSOL CONCENTRATION

 

Data on gravimetric aerosol concentrations are presented in the following table:

 

Group	Gravimetrically Determined Aerosol Concentration [mg/m^3air]	Target Aerosol Concentration [mg/m^3air]	Achieved from Target [%]
2	2.85 ± 0.42 (n=10, CV=14.6%)	3	95.1%
3	13.8 ± 1.3 (n=10, CV=9.5%)	15	92.0%
4*	82.1 ± 9.9 (n=3, CV=12.1%)	75	109.5%
	47.1 ± 4.8 (n=7, CV=10.3%)	45	104.8%

*         Data for group 4 were split for the periods with different target concentrations.

 

PARTICLE SIZE DISTRIBUTION

 

Data on particle size distribution are presented in the following table:

 

Group	Mean MMAD [µm] and (Mean GSD)	Range of MMAD [µm]	Range of GSD	Number of Determinations	Mean Percentage of Particles <3 µm [%]
2	1.58 (2.53)	1.41 - 1.80	2.25 - 2.90	3	75.5%
3	1.80 (3.20)	1.62 - 2.14	2.89 - 3.57	3	67.0%
4	2.93 (2.91)	2.66 - 3.18	2.70 - 3.15	4	50.9%

 

EXPOSURE CONDITIONS

 

The group means for temperature, relative humidity and oxygen concentration measured during the exposures were as follows:

 

Group	Temperature [°C]	Relative Humidity [%]	Oxygen Concentration [%]
1	23.3 ± 0.1 (n=10)	1.4 ± 0.0 (n=10)	20.2 ± 0.0 (n=10)
2	23.4 ± 0.1 (n=10)	1.2 ± 0.1 (n=10)	20.2 ± 0.0 (n=10)
3	23.1 ± 0.0 (n=10)	1.8 ± 0.0 (n=10)	20.4 ± 0.0 (n=10)
4	22.8 ± 0.0 (n=10)	0.5 ± 0.1 (n=10)	20.6 ± 0.0 (n=10)

Conclusions:

Wistar rats were exposed by nose-only flow past inhalation for 6 hours/day, 5 days/week over a period of two weeks. Control animals were exposed to air only. Exposure to gravimetrically determined aerosol concentrations of 2.85, 13.8, and 82.1 / 47.1 mg/m3 air was achieved in the different treatment groups and resulted in concentration-related focal squamous metaplasia with submucosal inflammation. In the mid-dose group, focal erosion or ulceration was additionally noted along with a reduced food intake and body weight gain. Further on to these findings, clinical signs (mainly effects on breathing, ruffled fur and salivation) were also noted in the high-dose group as well as increased neutrophil counts and histopathological findings in further respiratory tract organs and the death of one animal was recorded. Based on this incidence along with effects on breathing and body weight development, the aerosol concentration was reduced from 82.1 to 47.1 mg/m3 air.

The premature death of one low-dose animal was considered to be fortuitous as no cause of death could be established, in the absence of any effects in group 2 apart from minimal to mild histopathological findings and as neither mortalities nor clinical signs were recorded at the next higher concentration (13.8 mg/m3 air).

The test item was considered to cause concentration-related effects in the respiratory tract (mainly erosion, ulceration, squamous metaplasia and/or inflammation) that resulted in clinical signs (e.g. effects on breathing), increased neutrophil counts as an indicator of inflammation, reduced food intake and body weight gain as well as the death of one animal.

The increase in urea was not reflected in organ weight changes or histopathological findings and was therefore considered to be of no toxicological relevance.

Under the conditions of this study the upper and lower respiratory tract were considered to be target organs.

Based on the histopathological results, a no-observed-effect-level (NOEL) could not be established.

Possible target concentrations for a subsequent 90-day inhalation study are 0.1, 0.5 and 8 mg 4,4’-Dithiodimorpholine/m3 air.

Executive summary:

In this dose range finding study, 4,4’-Dithiodimorpholine was administered to four groups of 5 male and female Wistar rats by nose-only, flow-past inhalation for a period of 6 hours per day at 5 days/week for two weeks to target concentrations of 3, 15, and 75 mg/m3 air of 4,4’-Dithiodimorpholine in groups 2 to 4, respectively. Based on a premature death in group 4 after 2 days of treatment along with effects on breathing and body weight development at this concentration, the target aerosol concentration was reduced to 45 mg/m3 air from day 6. Animals of group 1 were exposed to air only. Throughout the study all animals were observed for viability, body weight, food consumption clinical signs, ophthalmoscopic findings and clinical laboratory investigations. Necropsy and macroscopic examination of all animals was performed and organ weights were recorded. Microscopic examination was performed thereafter.

 

TECHNICAL DATA

 

Gravimetrically determined aerosol concentrations of 2.85, 13.8 and 82.1 / 47.1 mg 4,4’-Dithiodimorpholine/m3 air were achieved in groups 2 to 4, respectively, which were within 92.0% and 109.5% of the respective targets.

 

The particles of the aerosol were considered to be respirable for rats, with gravimetrically determined mean Mass Median Aerodynamic Diameters between 1.58 and 2.93 µm.

 

The generated aerosols were considered to be acceptable for inhalation toxicity testing in rats and temperature, relative humidity and oxygen concentration during exposure were considered to be suitable for this type of study.

 

IN-LIFE DATA

 

One female exposed to 82.1 mg/m3 air (group 4) was found dead after 2 days of treatment. Death was attributed to severe inflammation and marked erosion/ulceration of the larynx. There were no further deaths that were considered to be related to treatment.

 

Clinical signs were limited to group 4 and were observed predominantly in males. They comprised mainly of effects on breathing (labored breathing, breathing noises and/or tachpynea), ruffled fur and salivation.

 

A concentration-related decrease in food intake was noted in groups 3 and 4.

 

Body weight loss was noted in group 4 from day 1 to day 4 of treatment. Thereafter, the animals generally gained weight again. A concentration-related reduced body weight gain was also noted for group 3.

 

Neutrophil counts were increased in females group 4.

 

POST MORTEM DATA

 

Focal squamous metaplasia with associated submucosal inflammation was recorded in the larynx of all groups with a concentration-related incidence and severity. These findings were accompanied by focal erosion, ulceration and/or focal epithelial hyperplasia.

 

Foreign-body granulomas were noted within the airways of the lungs of several group 4 animals and in a control female. These granulomata appeared to have caused no damage to the adjacent lung tissue.

 

Focal mucosal inflammation in the nasal cavity was observed in several group 4 animals. This finding was occasionally associated with focal degeneration and/or focal hyperplasia of the squamous epithelium.

 

Focal squamous metaplasia, submucosal inflammatory cell infiltration and/or focal ulceration were present in the pharynx of single animals.

 

There were no further relevant findings related to treatment with 4,4’-Dithiodimorpholine.

 

CONCLUSION

 

Under the conditions of this study the upper and lower respiratory tract were considered to be target organs.

 

Based on the histopathological results, a no-observed-effect-level (NOEL) could not be established.

 

Possible target concentrations for a subsequent 90-day inhalation study are 0.1, 0.5 and 8 mg 4,4’-Dithiodimorpholine/m3 air.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

0.507 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Sieber's study is considered to be a reliable study with a klimisch score of 1.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Key study : 90 -day repeated toxicity study by inhalation (Sieber 2014) 

The purpose of this inhalation study was to assess the cumulative toxicity of 4,4’-Dithiodimorpholine when administered 6 hours daily (5 days per week) to rats by nose-only, flow-past inhalation exposure for a period of 13 weeks. The reversibility or progression of any test item related effects or any delayed toxicity was assessed during a 6-week exposure free recovery period. This study was designed to indicate potential target organs and to provide a rational basis for the assessment of the toxicological risk to man. Groups of 10 male and 10 female Wistar rats each were exposed by nose-only flow-past inhalation to target concentrations of 0.10, 0.50 and 8.0 mg 4,4’-Dithiodimorpholine/m3 in each of groups 2 to 4, respectively. The rats of the control group were exposed to filtered air only (group 1). A additional rats of each sex in all groups were kept for a 6-week recovery period. Mortality, clinical signs, food consumption, body weights, ophthalmoscopic findings, clinical laboratory investigations, organ weights, macroscopic and microscopic findings were recorded. 

Exposure to gravimetrically determined atmosphere concentrations of 0.099, 0.507 and 8.38 mg 4,4’-Dithiodimorpholine/m3 air were achieved in groups 2 to 4, respectively, and were close to the respective targets. The chemically determined mean aerosol concentrations confirmed the gravimetric determinations. Temperature, relative humidity and oxygen concentration during exposure were considered to be suitable for this type of study. The mean MMAD (mean mass aerodynamic diameter) and GSD (geometric standard deviation) were 1.86 µm (GSD = 2.92) for group 3 and 1.89 µm (GSD = 2.36) for group 4. In conclusion, the test item was considered to be respirable to rats.

 A decreased body weight gain was noted in both sexes of groups 3 and 4 during the treatment period. In males, it was limited to the first 2 weeks of treatment. Increased body weight gain was noted in males and females of group 4 during the recovery period. Relative kidney weight was slightly but statistically significantly increased in females of group 3 and in males of group 4 when compared with controls. In the absence of any histopathologic findings in the kidneys, this finding is considered of no toxicological relevance. Histopathological changes in the larynx of both sexes were noted in a concentration-related manner. These findings included focal squamous metaplasia, focal hyperplasia of squamous epithelium, submucosal inflammation and focal erosion/ulceration. The latter finding was noted in group 4 only. After the end of the recovery period, complete recovery was observed in groups 2 and 3 and partial recovery in group 4.

There were no further findings that were considered to be related to treatment.

 Based on the findings in the larynx, the NOAEC (No-Observed-Adverse-Effect-Concentration) for local effects was considered to be 0.507 mg 4,4’-Dithiodimorpholine/m3 air. The NOAEC for systemic effects was considered to be 8.38 mg 4,4’-Dithiodimorpholine/m3 air.

First supporting study :28-day repeated toxicity study by inhalation (Ulrich 1987) 

In a study comparable to the OECD guideline # 412, four groups of young adult male and female rats (10/sex/dose) were exposed whole-body to 0, 1.1, 9.8 or 29 mg/m3 of sulfasan-R (4,4’dithiodimorpholine), as a fine dust aerosol, 6-hours per day, 5 day per week for 4 consecutive weeks. Observations were made with respect to the incidence of mortality, reactions displayed and body weight effects. Hematologic and clinical chemistry studies and urinalyses were conducted on all test and control animals on the day following final exposure. All animals were subjected to a gross necropsy. A complete set of organs and tissues were removed from each animal and preserved. Histopathologic studies were conducted on selected tissues and organs from the control and high-concentration groups. Weights of selected organs were recorded and subjected to statistical analyses. Minor signs of nasal irritation at 29 mg/m3 was the only pharmacotoxic signs observed. Body weights were markedly depressed for males at the high exposure level and were depressed to a lesser extent for high level females and mid level males. No exposure-related abnormalities were observed in ophthalmologic, hematologic, serum biochemical, urinalysis, or organ weight data. No toxicologically significant macroscopic abnormalities were observed at necropsy from any of the groups. Microscopic evaluation of various tissues from control animals, and animals exposed to 29 mg/m3 did not demonstrate any exposure-related abnormalities. Based on red discharge and decreased of body weight at 29 mg/m3 air in males, a NOAEC of 9.8 mg/m3 air was established in this study.

Second supporting study :14-day repeated toxicity study by inhalation (Schuler 2013) 

In this dose range finding study, 4,4’-Dithiodimorpholine was administered to four groups of 5 male and female Wistar rats by nose-only, flow-past inhalation for a period of 6 hours per day at 5 days/week for two weeks to target concentrations of 3, 15, and 75 mg/m3 air of 4,4’-Dithiodimorpholine in groups 2 to 4, respectively. Based on a premature death in group 4 after 2 days of treatment along with effects on breathing and body weight development at this concentration, the target aerosol concentration was reduced to 45 mg/m3 air from day 6. Animals of group 1 were exposed to air only. Throughout the study all animals were observed for viability, body weight, food consumption clinical signs, ophthalmoscopic findings and clinical laboratory investigations. Necropsy and macroscopic examination of all animals was performed and organ weights were recorded. Microscopic examination was performed thereafter.

Gravimetrically determined aerosol concentrations of 2.85, 13.8 and 82.1 / 47.1 mg 4,4’-Dithiodimorpholine/m3 air were achieved in groups 2 to 4, respectively, which were within 92.0% and 109.5% of the respective targets. The particles of the aerosol were considered to be respirable for rats, with gravimetrically determined mean Mass Median Aerodynamic Diameters between 1.58 and 2.93 µm. The generated aerosols were considered to be acceptable for inhalation toxicity testing in rats and temperature, relative humidity and oxygen concentration during exposure were considered to be suitable for this type of study.

One female exposed to 82.1 mg/m3 air (group 4) was found dead after 2 days of treatment. Death was attributed to severe inflammation and marked erosion/ulceration of the larynx. There were no further deaths that were considered to be related to treatment. Clinical signs were limited to group 4 and were observed predominantly in males. They comprised mainly of effects on breathing (labored breathing, breathing noises and/or tachpynea), ruffled fur and salivation. A concentration-related decrease in food intake was noted in groups 3 and 4. Body weight loss was noted in group 4 from day 1 to day 4 of treatment. Thereafter, the animals generally gained weight again. A concentration-related reduced body weight gain was also noted for group 3. Neutrophil counts were increased in females group 4.

Focal squamous metaplasia with associated submucosal inflammation was recorded in the larynx of all groups with a concentration-related incidence and severity. These findings were accompanied by focal erosion, ulceration and/or focal epithelial hyperplasia. Foreign-body granulomas were noted within the airways of the lungs of several group 4 animals and in a control female. These granulomata appeared to have caused no damage to the adjacent lung tissue. Focal mucosal inflammation in the nasal cavity was observed in several group 4 animals. This finding was occasionally associated with focal degeneration and/or focal hyperplasia of the squamous epithelium. Focal squamous metaplasia, submucosal inflammatory cell infiltration and/or focal ulceration were present in the pharynx of single animals. 

There were no further relevant findings related to treatment with 4,4’-Dithiodimorpholine. 

Under the conditions of this study the upper and lower respiratory tract were considered to be target organs. Based on the histopathological results, a no-observed-effect-level (NOEL) could not be established.

Justification for classification or non-classification

No classification for repeated toxicity is required for DTDM based on the 90 -day repeated toxicity study by inhalation according to the Regulation (EC) No 1272/2008.