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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-11-12 to 2019-09-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2018-11-30 to 2018-12-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline available
Principles of method if other than guideline:
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for 28 days at dose levels of 25, 75 and 200 mg/kg bw/day.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany (females) or Charles River Laboratories France, L'Arbresle Cedex, France.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6-8 weeks
- Weight at study initiation: 153 to 242 g (females), 100 to 300 g (males)
- Housing: up to 5 animals of same sex and same dosing group
- Diet: ad libitum
- Water (e.g. ad libitum): tap water via water bottles
- Acclimation period: for at least 5 days before the commencement of dosing

DETAILS OF FOOD AND WATER QUALITY: The feed is analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Test Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
Municipal tap water will be freely available to each animal via water bottles. Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility.
It is considered that there are no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 °C
- Humidity (%): 40-70 %
- Air changes (per hr): at least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hours light and 12-hours dark
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
Polyethylene glycol 400, specific gravity 1.125
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle was continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item. Any residual volumes were discarded.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
3/sex/dose
Control animals:
no
Details on study design:
- Dose selection rationale: The dose levels were selected at request of the Sponsor based on the results of two acute oral toxicity studies in rats provided by the Sponsor. In the first study the LD50 for diallyl 2,2'-oxydiethyl dicarbonate was calculated to be 416 mg/kg for males, and greater than 400 mg/kg and less than 600 mg/kg for females and 515 mg/kg for both sexes. Subsequently a second acute toxicity study was performed where the LD50 value was 349 mg/kg bw.
- Rationale for animal assignment (if not random): Animals were assigned to groups by a stratified randomisation scheme designed to achieve similar group mean body weights, with all animals within ± 20% of the sex mean. Males and females were randomised separately. Animals in poor health or at extremes of body weight range were not assigned to groups.
- Fasting period before blood sampling for clinical biochemistry: Animals will be fasted (overnight with a maximum of 24 hours) before blood sampling, but water will be available.
Positive control:
no positive control
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least three times daily up to de day prior to necropsy, 0, 1 and 3 hours after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly, starting Day 1, throughout the dosing period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No feeding study, but food consumption monitored twice weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood will be collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood will be collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane.
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.2] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: COAGULATION
- Time schedule for collection of blood: Blood will be collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane.
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.3] were examined.

Sacrifice and pathology:
All animals were subjected to an external, thoracic and abdominal examination on Day 29 (scheduled necropsy) or sooner (decedents). Animals were deprived of food prior to necropsy. The organs identified for weighing were weighed at necropsy for all scheduled euthanasia animals. Organs were not fixed and histopathological examination was not performed.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation (all treatment groups) and piloerection was observed (highest dose group).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female (No. 17) at 200 mg/kg/day was found dead on Day 25. Because of minimal clinical signs, the cause of death was expected not to be related to treatment with the test item.
Body weight and weight changes:
no effects observed
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:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Alanine-aminotransferase activity was increased in individual animal No. 7 and 18 (highest dose group), but overall normal range
Urinalysis findings:
not examined
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:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Remarks on result:
not measured/tested
Conclusions:
Based on the results of the dose range finder, selected dose levels of diallyl 2,2'- oxydiethyl dicarbonate for the 90-day toxicity study were 25, 100 and 300 mg/kg.
The peak effect of occurrence of clinical signs occurred at 0-1 hours after dosing. This time point was taken into account to set a time range within which clinical observations and functional observation tests were conducted after dosing in the main study.
Executive summary:

A dose range finding study in rats was conducted to select dose levels of diallyl 2,2'- oxydiethyl dicarbonate for the 90 -day toxicity study and to determine the peak effect of occurrence of clinical signs after dosing. No testing guidelines are applicable as this dose range finder is intended for dose level selection purposes only.

Male and female rats (3/sex/dose) were orally exposed to 25, 75 or 200 mg/kg bw/day of the test item via gavage. Minimal clinical signs (salivation) were observed in all dose groups, while no treatment-related effects on mortality, body weights, food consumption, hematology, coagulation, clinical chemistry and organs (weights and macroscopic examination) were found. Based on the results of the dose range finder, selected dose levels for the main study were 25, 100 and 300 mg/kg.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Diallyl 2,2'-oxydiethyl dicarbonate
EC Number:
205-528-7
EC Name:
Diallyl 2,2'-oxydiethyl dicarbonate
Cas Number:
142-22-3
Molecular formula:
C12H18O7
IUPAC Name:
3-({[2-(2-{[(prop-2-en-1-yloxy)carbonyl]oxy}ethoxy)ethoxy]carbonyl}oxy)prop-1-ene
Test material form:
liquid
Remarks:
clear, colorless

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6-8 weeks
- Weight at study initiation: 100 to 200 g (females), 100 to 300 g (males)
- Fasting period before study:
- Housing: up to 5 animals of same sex and same dosing group
- Diet: ad libitum
- Water (e.g. ad libitum): tap water via water bottles
- Acclimation period: for at least 5 days before the commencement of dosing

DETAILS OF FOOD AND WATER QUALITY:
The feed is analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Test Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
Municipal tap water will be freely available to each animal via water bottles. Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there are no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 °C
- Humidity (%): 40-70 %
- Air changes (per hr): at least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hours light and 12-hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
Polyethylene glycol 400, specific gravity 1.125
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle was continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item. Any residual volumes were discarded.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. Trial preparation formulations will not be used for dosing and will be discarded after the assessment is complete. These trial preparations have a non-GLP status and will be carried out in the quality assured environment of the Test Facility.
- Concentration in vehicle: 0, 6, 20, 60/40 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for Analysis: Duplicate middle samples for Groups 1 and 3 (concentration analysis only) and duplicate top, middle, and bottom samples for Groups 2 and 4 (concentration and homogeneity analysis).
Sample Volume: Approximately 500 mg accurately weighed.
Acceptance Criteria: Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ± 10%.

Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Top dose changed from 300 mg/kg bw/day after 3 days of dosing due to high mortality rate
No. of animals per sex per dose:
15/sex/dose (control), 10/sex/dose (low and mid dose groups), 17 (females) or 18 (males) per dose (high dose group, as animals found dead on Day 3 were replaced by 2 female and 3 male spare animals)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were amended based on the results of the dose range finding study (see cross reference)
- Rationale for animal assignment (if not random): Animals were assigned to groups by a stratified randomisation scheme designed to achieve similar group mean body weights, with all animals within ± 20% of the sex mean. Males and females were randomised separately. Animals in poor health or at extremes of body weight range were not assigned to groups.
- Fasting period before blood sampling for clinical biochemistry: Animals will be fasted (overnight with a maximum of 24 hours) before blood sampling, but water will be available.
Positive control:
no positive control

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily, starting during the pretreatment period and throughout the dosing period; 0-1 hour postdose during the dosing period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly, starting during the pretreatment period, and on the day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly, starting Day 1 (at predose). A fasted weight was recorded on the day of necropsy. In order to monitor the health status, Animal No. 42 was also weighed on Day 70, Animal No. 100 was also weighed on Day 84 and Animal No. 98 was also weighed on Day 87.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No feeding study
- Food consumption for each animal determined: Yes (quantitatively measured except for day of scheduled euthanasia)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes, but no drinking water study
- Procedure: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Examination during Pretreatment in all animals (including spare animals), and at the end of the Dosing Period in Week 13 in all Group 1 and 4 animals.
- Procedure: The eyes were examined using an ophthalmoscope after application of a mydriatic agent (Tropicol 5 mg/ml solution)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment (all animals)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: all surviving animals
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment (all animals)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: all surviving animals
- Parameters checked in table [No.1] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: end of treatment (all animals)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once at week 12-13 of treatment
- Dose groups that were examined: all
- Battery of functions tested: locomotor activity, hearing ability, fore- and hind-limb grip strength

IMMUNOLOGY: No

OTHER:
ARENA OBSERVATIONS
Once before the first administration of the test item and at weekly intervals during the treatment period.

ESTROUS STAGE DETERMINATION

THYROID HORMONE ANALYSES
- Serum samples at a target volume of 1.0 mL were collected in tubes without anticoagulant.

SPERM ANALYSES
- Sperm motility: All surviving males at necropsy.
- Sperm morphology: Males of the control and the high dose group at necropsy.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3)

HISTOPATHOLOGY: Yes (see table 4)
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels, unless otherwise noted. Pairwise comparisons were made for each of Group 2, 3 or 4 vs. Group 1.
Statistics for data collected in Provantis
Analyses for the variables bw, bw gains, food cons. and organ weights were performed as follows when possible, but exclude any group with less than 3 observations.
Levene’s test were used to assess the homogeneity of group variances. Groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or Kruskal-Wallis test if it was significant. If overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test.
The data corresponding to a response variable of interest and to a related covariate were submitted to an analysis of covariance, including only groups with at least three non-missing paired values and if found to be significant, then pairwise comparisons were conducted using Dunnett’s test.

Statistics for Clin. path,; arena observ., funct. observ.
Inferential statistics were performed for pairwise comparisons, but exclude semi-quantitative data and any group with less than 2 observations.
Parametric
Datasets with at least 3 groups (control group, 2 other groups) were compared using Dunnett-test.
For motor activity (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.
Non-Parametric
Datasets with at least 3 groups were compared using Steel-test.
Incidence
An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test was significant.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The following clinical signs were observed in the animals dead or sacrificed on Days 9 to 87: salivation, hunched posture, erected fur, eyes partly closed, liquid feces, increased or decreased activity and or weakness. In the surviving animals at 300/200 mg/kg/day, salivation, liquid feces and/or ploughing were observed during the dosing period.
Any other clinical sign noted during the Dosing Period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:
mortality observed, treatment-related
Description (incidence):
At the high dose level 10 males and 7 females were found dead or send to necropsy because of severe clinical signs between Days 3 and 87. In addition, one male (No. 16) at 25 mg/kg/day was found dead on Day 36. In this animal pale fluid was observed in the body cavity and the lung failed to collapse, probably due to a gavage trauma. Deaths at dosing are considered to be of incidental nature and not test item-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight gains of treated animals at 25 and 100 mg/kg/day remained in the same range as controls over the study period.
A test item-related lower body weight (gain) was observed in males at 300/200 mg/kg/day compared to controls from Day 8 onwards, reaching statistical significance from Day 22 onwards for body weights. Mean body weight and body weight gain were 0.87x and 0.78x of controls, respectively at the end of the Dosing Period.
A significantly higher body weight (gain) was observed in females at 300/200 mg/kg/day compared to control on several occasions from Day 1 onwards. Mean body weight was 1.09x of controls at the end of the Dosing Period. However, body weights stayed within the range considered normal for rats of this age and strain and the opposite effect would be expected in case of toxicity. These findings were therefore considered to be unrelated to treatment with the test item.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption of treated animals at 25 and 100 mg/kg/day remained in the same range as controls over the study period.
At 300/200 mg/kg/day, a lower food consumption compared to control animals was observed in both males (0.83x, 0.81x and 0.84x of controls over Days 1-4, Days 4-8 and Days 8-11, respectively) and females (0.78x of controls over Days 1-4). On several occasions thereafter, a higher food consumption compared to control animals was observed in females at 300/200 mg/kg/day (1.10x, 1.12x, 1.12x, 1.12x, 1.14x of controls over Days 8-11, Days 11-15, Days 29-36, Days 43-50 and Days 57-64, respectively). However, food consumption stayed within the range considered normal for rats of this age and strain and the opposite effect would be expected in case of toxicity. These findings were therefore considered to be unrelated to treatment with the test item.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopy parameters were not affected by treatment with the test item.
The nature and incidence of ophthalmology findings noted during the Pretreatment Period and in Week 13 were similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At 25 mg/kg/day no test item related effects on hematology were observed.
A slight decrease in mean corpuscular volume, mean corpuscular haemoglobin was observed in males and females at 100 and 300/200 mg/kg/day, and a slight increase in white blood cells, platelet count and red cell distribution width was found in males and females at 300/200 mg/kg/day. However, as the changes in MCV, MCH, WBC, RDW and platelets remained within the normal range of rats of this age and strain, these were considered of no biological relevance. Moreover, the slightly decreased haemoglobin and haematocrit levels observed in males at 300/200 mg/kg/day were considered to be of no toxicological significance due to the absence of correlated microscopic findings and the minimal magnitude of the change.
Furthermore, an increased reticulocyte count was observed in females at 300/200 mg/kg/day, however this increase was mainly due to the extreme high value (6.65x of control mean) of a single animal (Female No. 90). The differences in most other red blood cell parameters (RDW, haemoglobin, haematocrit, MCV, MCHC) were also mainly caused by the results of this single animal (see tabular presentation of results attached below).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No test item-related changes in clinical chemistry parameters were noted at 25 mg/kg/day.
The following statistically significant changes were noted in treated males and/or females at 100 and/or 300/200 mg/kg/day:
An increase was observed in alanine-aminotransferase and aspartate-aminotransferase activity in males and females at 300/200 mg/kg/day. Moreover, a slight increase was observed in alkaline phosphatase activity in mles starting at 100 mg/kg/day and High Density lipoprotein (HDL), Low Density lipoptrotein (LDL) and total cholesterol levels in males and females at 300/200 mg/kg/day. However, the slightly increased HDL, LDL and total cholesterol levels observed in males and females were considered test item related but non-adverse at this incidence and severity.
A slight increase in urea levels was observed in males at 100 and 300/200 mg/kg/day. Above this, bilirubin levels were increased in males and females at 300/200 mg/kg/day.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No test item-related changes in urine parameters were noted in treated animals.
While few changes were statistically significant, the alterations in urinalysis parameters were unrelated to administration of the test item due to the minimal magnitude of the change.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related organ weight differences were noted in treated animals starting at 100 mg/kg/day in males and at 300/200 mg/kg/day in females. Results are summarised in the tables attached below.
Higher mean liver weight was noted at 300/200 mg/kg/day in males (relative to body weight only) and females (as absolute value and relative to body weight). At 100 mg/kg/day in males higher mean liver weight was generally of low magnitude but was statistically significant expressed relative to body weight. Higher mean spleen weight was noted in males and females at 300/200 mg/kg/day (as absolute value and relative to body weight).
For the following changes in organ weights found in animals treated with 300/200 mg/kg/day, no macroscopic or microscopic correlate was observed: lower mean thymus weight in males (absolute value only) and females (relative to body weight only), higher mean kidney weight in males (relative to body weight only) and in females (absolute and relative to body weight), and higher mean heart weight in males (relative to body weight only) and in females (absolute value only).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related macroscopic findings were observed in males and females starting at 100 mg/kg/day (liver and stomach) and at 300/200 mg/kg/day (spleen, pancreas, duodenum and adrenal glands). Results are summarised in the tables attached below.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with Diallyl 2,2’-oxydiethyl dicarbonate were noted in males and females starting at 100 mg/kg/day in the liver, stomach, and duodenum and at 300/200 mg/kg/day only in the spleen, mesenteric lymph node, pancreatic lymph nodes (examined as gross lesions) of males and females, and the jejunum of males only. Results are summarised in the tables attached below.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Sperm analysis revealed no treatment-related effect in the high dose group compared to concurrent control animals.
No test item-related changes in thyroid hormones were noted at 25 and 100 mg/kg/day. Moreover, serum levels of thyroid stimulating hormone (TSH) and Triiodothyronine (T3) were considered unaffected by treatment with the test item up to 300/200 mg/kg/day. The decreased total T4 levels in males and increased in T4 levels females at 300/200 mg/kg/day were considered to be of no toxicological significance due to the absence of correlated microscopic findings and the minimal magnitude of the change.
Details on results:
for tabular presentation of results please refer to 'Attached backround material'

Effect levels

Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 3: Summary Test Item-Related Macroscopic Findings - Main Study (Day 92/93)

 

Males

Females

Dose level (mg/kg/day)

0

25

100

200/ 300

0

25

100

200/ 300

LIVERa

15

9

10

8

15

10

10

10

Focus pale

-

-

1

6

-

-

2

6

Focus dark

-

-

1

4

-

-

-

1

Enlargement

-

-

-

2

-

-

-

5

Irregular surface

-

-

-

4

-

-

-

6

Small, left lateral lobe

-

-

-

-

-

-

-

1

Prominent lobular architecture

1

-

-

2

-

-

1

2

 

 

 

 

 

 

 

 

 

STOMACHa

15

9

10

8

15

10

10

10

Irregular surface (non-glandular mucosa)

-

-

1

3

-

-

6

3

 

 

 

 

 

 

 

 

 

SPLEEN

15

9

10

8

15

10

10

10

Enlargement

-

-

-

3

-

-

-

3

Irregular surface

-

-

-

2

-

-

-

-

 

 

 

 

 

 

 

 

 

PANCREASa

15

9

10

8

15

10

10

10

Nodule

-

-

-

3

-

-

-

5

 

 

 

 

 

 

 

 

 

SMALL INTESTINE, DUODENUM

15

9

10

8

15

10

10

10

Thick

-

-

-

1

-

-

-

3

Dilatation

-

-

-

-

-

-

-

1

 

 

 

 

 

 

 

 

 

a= Number of tissues examined from each group

Table 4: Summary Test Item-Related Microscopic Liver Findings

Scheduled Euthanasia Animals (Day 92/93)

 

Males

Females

Dose level (mg/kg/day)

0

25

100

300/ 200

0

25

100

300/ 200

LIVERa

10

9

10

8

10

10

10

10

NECROSIS; periportal

 

 

 

 

 

 

 

 

Minimal

-

-

-

1

-

-

-

-

Mild

-

-

-

-

-

-

-

4

Moderate

-

-

-

4

-

-

1

1

 

 

 

 

 

 

 

 

 

SINGLE CELL NECROSIS; hepatocellular, periportal

 

 

 

 

 

 

 

 

Minimal

-

-

1

1

-

-

-

1

Mild

-

-

1

2

-

-

1

1

 

 

 

 

 

 

 

 

 

HYPERPLASIA BILIARY

 

 

 

 

 

 

 

 

Minimal

-

-

1

-

-

-

1

1

Mild

-

-

2

2

-

-

-

5

Moderate

-

-

-

4

-

-

2

4

Marked

-

-

-

2

-

-

-

-

 

 

 

 

 

 

 

 

 

FIBROSIS; portal

 

 

 

 

 

 

 

 

Minimal

-

-

2

-

-

-

-

4

Mild

-

-

-

2

-

-

-

2

Moderate

-

-

-

1

-

-

1

-

Marked

-

-

-

2

-

-

-

-

 

 

 

 

 

 

 

 

 

INFILTRATION, MIXED CELL; portal

 

 

 

 

 

 

 

 

Minimal

-

-

-

-

-

-

1

2

Mild

-

-

-

4

-

-

1

1

 

 

 

 

 

 

 

 

 

PIGMENTED MACROPHAGE; portal

 

 

 

 

 

 

 

 

Minimal

-

-

2

-

-

-

1

5

Mild

-

-

2

6

-

-

1

2

 

 

 

 

 

 

 

 

 

HYPERTROPHY; hepatocellular, periportal

 

 

 

 

 

 

 

 

Minimal

-

-

4

1

-

-

5

4

Mild

-

-

2

7

-

-

2

6

 

 

 

 

 

 

 

 

 

INCREASED MITOSES; hepatocellular, periportal

 

 

 

 

 

 

 

 

Minimal

-

-

-

2

-

-

1

1

Mild

-

-

-

-

-

-

1

1

 

 

 

 

 

 

 

 

 

HYPERPLASIA; hepatocellular, regenerative

 

 

 

 

 

 

 

 

Minimal

-

-

-

-

-

-

-

1

Mild

-

-

-

3

-

-

-

-

 

 

 

 

 

 

 

 

 

VACUOLATION; hepatocellular, periportal

 

 

 

 

 

 

 

 

Minimal

2

2

4

8

1

1

5

6

Mild

-

-

-

-

-

-

1

2

a= Number of tissues examined from each group.

Applicant's summary and conclusion

Conclusions:
Based on the results of this subchronic repeated dose oral toxicity study with Diallyl 2,2’-oxydiethyl dicarbonate in rats, a NOAEL of 25 mg/kg bw/day could be derived for this substance.
Executive summary:

The subchronic oral toxicity of the test item Diallyl 2,2’-oxydiethyl dicarbonate was studied in rats in a 90-day repeated dose toxicity test according to OECD Test Guideline 408 under GLP.

In this study, the test item Diallyl 2,2’-oxydiethyl dicarbonate was orally administered to rats via gavage. Based on the 28-d dose range finding study, the dose levels 25, 100 and 300 mg/kg bw/day were selected. Due to unscheduled mortalities observed on Day 3 in the high dose group the high dose level was reduced to 200 mg/kg bw /day from Day 4 on.

The following parameters and endpoints were evaluated in this study: clinical signs, body weights, food consumption, ophthalmology, functional tests, estrous stage determination, clinical pathology parameters (hematology, coagulation, clinical chemistry, thyroid hormones and urinalysis), gross necropsy findings, organ weights, sperm analysis and histopathologic examinations.

At the high dose level 10 males and 7 females were found dead or send to necropsy because of severe clinical signs between Days 3 and 87. The following clinical signs were observed in these animals: salivation, hunched posture, erected fur, eyes partly closed, liquid feces, increased or decreased activity and/or weakness.

In all surviving males at 300/200 mg/kg/day, an adverse test-item related lower body weight (gain) compared to controls was observed from Day 8 onwards, correlating with lower food consumption. At clinical chemistry, bilirubin levels, alkaline phosphatase (ALP), alanine-aminotransferase (ALAT) and aspartate-aminotransferase (ASAT) activities were increased in males and/or females at 300/200 mg/kg/day.  

Test item-related organ weight differences were noted in the starting at 100 mg/kg/day in males and at 300/200 mg/kg/day in females. Furthermore, test item-related microscopic findings after treatment with Diallyl 2,2’-oxydiethyl dicarbonate were noted in males and females starting at 100 mg/kg/day in the liver, stomach, and duodenum and at 300/200 mg/kg/day only in the spleen, mesenteric lymph node, pancreatic lymph nodes (examined as gross lesions) of males and females, and the jejunum of males only. However, only the histopathological findings in the liver were considered adverse.

Sperm analyses revealed no treatment-related effects. Furthermore, no treatment-related effects on urine parameters, ophthalmoscopy or functional parameters were detected. No test item-related changes in thyroid hormones were noted at 25 and 100 mg/kg/day. Moreover, serum levels of thyroid stimulating hormone (TSH) and Triiodothyronine (T3) were considered unaffected by treatment with the test item up to 300/200 mg/kg/day. The decreased total T4 levels in males and increase in T4 levels in females at 300/200 mg/kg/day were considered to be of no toxicological significance due to the absence of correlated microscopic findings and the minimal magnitude of the change. 

In conclusion, administration of Diallyl 2,2’-oxydiethyl dicarbonate by once daily oral gavage was well tolerated in rats at a dose level of 25 mg/kg/day. Test item-related preterminal deaths were present at 300/200 mg/kg/day. Adverse effects were observed at levels of 100 and 300/200 mg/kg/day and consisted of coagulative necrosis, single cell necrosis, biliary hyperplasia, and portal fibrosis of the liver, with correlating macroscopic findings, organ weights and clinical pathology changes. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 25 mg/kg/day.