Diallyl 2,2'-oxydiethyl dicarbonate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 APR 2020 to 27 MAY 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Reference

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

Reason / purpose for cross-reference:

reference to other study

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

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

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

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

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.

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'

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

Critical effects observed:

not specified

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
LIVER^a	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
STOMACH^a	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	-	-	-	-
PANCREAS^a	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
LIVER^a	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.

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.

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

developmental toxicity

Remarks:

Dose-range finder

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

13 Apr 2020 to 28 Apr 2020 (initiating of dosing and completion of in-life phase)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

This study was used for dose level selection purposes only.

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Guideline:

other: No guidelines are applicable as this study was used for dose level selection purposes only. If not mentioned otherwise, test system, procedures and techniques were identical to those used during the Main study

Principles of method if other than guideline:

- Purpose of the test: Selection of dose levels for the main study.
- Short description of test conditions: Two groups of 6 females were exposed to 0 and 200 mg/kg/day for Days 6 to 20 post-coitum inclusive by oral gavage
- Parameters analysed / observed:
Maternal: mortality/moribundity, clinical observations, body weights, food consumption, water consumption, necropsy (uterine examination), organ weights
Fetal: litter sizes, sex ratio fetal, body weights, external examination

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

time-mated female Wistar Han Rats

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 11-15 weeks
- Weight at study initiation: 205 to 246 g

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C (mean value)
- Humidity (%): 44 to 52%

Note: Husbandry identical as for the main study

IN-LIFE DATES: From: 07 Apr 2020 (animal arrival) To: 28 Apr 2020 (necropsy)

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

400

Details on exposure:

VEHICLE
- Concentration in vehicle: 0, and 40 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Samples for dose formulation analysis were not collected during the dose range finder, as concentration, homogeneity, and stability analysis was not performed.
However, to limit the impact, the test item preparation was performed with approved procedures and documented in detail. Preparations were visually inspected for homogeneity prior to use and all preparations were used within 24 hours after preparation of the formulation.

Details on mating procedure:

The females arrived on Day 0 post-coitum (Day 0 post-coitum is defined as the day of successful mating).

Duration of treatment / exposure:

14 Days, from Day 6 to Day 20 post-coitum, inclusive

Frequency of treatment:

Once daily, 7 days a week

Duration of test:

14 Days

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Dose / conc.:

200 mg/kg bw/day

No. of animals per sex per dose:

6 females/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose level was selected based on the results of a 90-day repeated dose toxicity study with oral gavage administration of diallyl 2,2’-oxydiethyl dicarbonate in rats and based on the results of a reproduction/developmental toxicity screening test. Based on the results of the above-mentioned studies, it was decided to use 200 mg/kg/day as dose level for the dose range finding study.

Maternal examinations:

- All animals (including Female No. 7 that was found dead on the day of scheduled necropsy) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded.
- The liver, stomach and macroscopic abnormalities were collected from all animals.
- The uterus and liver were weighed at necropsy for all scheduled euthanasia animals.
- Organ weight as a percent of body weight (using the body weight on Day 21 post-coitum) was calculated.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantation sites: Yes
- The number and distribution of live and dead fetuses: Yes
- The number and distribution of early and late resorptions: Yes
- The sex of each fetus based on the anogenital distance (not for fetuses of Female No. 7).
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- Each viable fetus of animals surviving to planned necropsy was externally examined in detail, weighed and sexed.
- All live fetuses were euthanized by decapitation.
- Fetuses of animals found dead before planned necropsy were externally examined in detail and euthanized by decapitation.
- No visceral (internal) or skeletal examination was performed

Clinical signs:

no effects observed

Description (incidence and severity):

No toxicologically relevant clinical signs were noted at 200 mg/kg/day.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Mortality was observed in one animal only. The cause for this death was not determined within this dose range finding study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight, body weight gain and corrected body weight were comparable between animals treated at 200 mg/kg/day and the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was comparable between animals treated at 200 mg/kg/day and the control group

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg/day, macroscopic observations at necropsy revealed two females with gray-white foci on the liver and two females with an enlarged liver. The thyroid gland of one of these females was found to be pale as well and on the thymus dark red foci were present.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

not examined

Pre- and post-implantation loss:

not examined

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

not examined

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Other effects:

no effects observed

Description (incidence and severity):

All females were found to be pregnant with viable fetuses.

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal body weights were within normal values for male and female fetuses at the 200 mg/kg/day dose group.

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratios were within normal values

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Litter sizes were within normal limits for all groups

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

External examination of the fetuses did not show any abnormalities

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

not examined

Abnormalities:

no effects observed

Conclusions:

In conclusion, no toxicological relevant changes in reproductive and developmental parameters were observed and there were no adverse effects on clinical signs, body weight, body weight gain, food consumption of females. Treatment-related macroscopic findings on the liver at 200 mg/kg bw/day were observed, furthermore one death occurred in this dose group (cause of death undetermined). Therefore, the selected dose levels for the Main study were 50, 100 and 200 mg/kg/day to aim for some toxic effects without excessive lethality at the top dose.

Executive summary:

A dose range finder was conducted to select dose levels for the OECD 414. No guidelines are applicable as this study was used for dose level selection purposes only. In this study Diallyl 2,2’-oxydiethyl dicarbonate was orally administered daily via gavage to pregnant rats from gestation day (GD) 6 to 20, at dose levels of 0 or 200 mg/kg/day. Subsequently, some parameters on systemic, reproductive and developmental toxicity were analysed.

All females were found to be pregnant with viable fetuses. No test item-related changes for the number of postimplantation loss were observed. One female was found dead on the day of scheduled necropsy (with enlarged pale liver with an irregular surface and gray-white foci; pale thyroid gland; dark red foci in the thymus). No toxicologically relevant clinical signs were noted at 200 mg/kg/day. Body weight, body weight gain and corrected body weight and food consumption were comparable between treated animals and control animals. At 200 mg/kg/day, macroscopic observations at necropsy revealed two females with gray-white foci on the liver and two females with an enlarged liver. In addition, the thyroid gland of one of these females was found to be pale. Litter sizes, sex ratio, fetal body weights, and external examination were within normal limits for all groups.

In conclusion, no toxicological relevant changes in reproductive and developmental parameters were observed. Furthermore all females were pregnant with viable foetus with no adverse effects on clinical signs, body weight, body weight gain, food consumption were observed. Treatment-related macroscopic findings on the liver at 200 mg/kg bw/day were overserved and one unexplained death occurred in this dose group. Therefore, the selected dose levels for the Main study were 50, 100 and 200 mg/kg/day to aim for some toxic effects without excessive lethality at the top dose.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

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

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability for at least 24 hours at room temperature under normal laboratory light conditions and for at least 8 days in the refrigerator is confirmed over the concentration range 0.889 mg/g (1 mg/mL) to 177 mg/g (200 mg/mL) (solutions)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

female Wistar Han Rats

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks old
- Weight at study initiation: 182 to 270 g
- Housing: Housed individually, in plastic cages containing appropriate bedding
- Diet (e.g. ad libitum): Pelleted rodent diet, provided ad libitum throughout the study, except during designated procedures
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 20°C
- Humidity (%): 50 to 86 % (The values that were outside the targeted range occurred for 11 out of 22 days with a mean relative humidity of 72 to 86% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the
study.)
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

IN-LIFE DATES: From: 16/17 JUN 2020 (animal receipt) To: 09 JUL 2020 (Last date of necropsy)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

Polyethylene glycol 400

Details on 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 at least weekly, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator.

VEHICLE
- Concentration in vehicle: 0, 10, 20, 40 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose Formulation Sample Collection Schedule:
- Occasion: Week 1 of treatment
- Concentration: All groups (The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results.)
- Homogeneity: Groups 2 and 4 (The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results.)

All samples to be analyzed were transferred (at room temperature under normal laboratory light conditions) to the analytical laboratory at the Test Facility. Analyses were performed using a validated analytical procedure.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ±10% of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%.

Formulation analyses confirmed that formulations of the test item in polyethylene glycol 400 were prepared accurately and homogenously.

Details on mating procedure:

Time-mated females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating)

Duration of treatment / exposure:

Day 6 to Day 20 post-coitum, inclusive

Frequency of treatment:

Once daily oral gavage 7 days a week

Duration of test:

Day 6 post-coitum - scheduled necopsy: Day 21 post-coitum

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

200 mg/kg bw/day

No. of animals per sex per dose:

22 females/ group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected based on the results of the dose range finder in the rat and the results of the 90-Day study in an attempt to produce graded responses to the test item.

- Other:
Justification of Route: The oral route of exposure was selected because this is the most appropriate route of administration for this substance for the assessment of reproductive toxicity and this was specified in the final decision by ECHA.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Animals were observed for general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily, beginning on Day 2 post-coitum onwards up to the day prior to necropsy

BODY WEIGHT: Yes (weighed individually)
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum (The body weight of the animals of Subgroup 4 were performed on Day 3 post-coitum instead of Day 2 post-coitum. Body weight recorded for Subgroup 4 on Day 3 instead of Day 2 post-coitum were excluded from the data tables and were reported in the key to missing values. As dosing was only initiated on Day 6 post-coitum no data on possible test item effects are missing and based on the data of the other subgroups, sufficient information was available for evaluation).
Female No. 70 was also weighed on Day 11 post-coitum (in order to monitor the health status).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.
Food consumption of the animals of subgroup 4 were performed on Day 3 post-coitum instead of Day 2 post-coitum. Food consumption data recorded for subgroup 4 on Day 3 instead of Day 2 post-coitum were excluded from the data tables and were reported in the key to missing values. As dosing was only initiated on Day 6 post-coitum no data on possible test item effects are missing and based on the data of the other subgroups, sufficient information was available for evaluation.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21 post-coitum
Organ Weights: Liver, thyroid gland, and uterus were weighed at necropsy (except for females that delivered their offspring early, organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis). Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of one of the organs of a pair were taken and entered as a tissue comment. Organ to body weight ratio (using the body weight on Day 21 post-coitum) were calculated.
Necropsy: External, thoracic and abdominal examination
Histology: Thyroid glands of all animals were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.
Histopathology: Gland, thyroid, liver, stomach
Clinical Pathology: Thyroid Hormone Parameters (Triiodothyronine (T3), Thyroxine (T4), Thyroid-Stimulating Hormone (TSH))

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gavid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- Blood was collected on the day of scheduled necropsy (Blood for thyroid hormone analysis was only collected from animals surviving until scheduled necropsy)
- Animals were not fasted overnight
- Volume collected: 1.0 mL

Fetal examinations:

- External examinations: Yes: Each viable fetus was sexed, examined in detail to detect macroscopic visible abnormalities and their weight (not determined for fetuses of animals found dead, or sacrificed before planned necropsy)
- Soft tissue examinations (visceral): Yes: (one-half of the fetuses (live and dead) in each litter (all groups))
- Skeletal examinations: Yes: (one-half of the fetuses (i.e. the fetuses with heads)
- Head examinations: Yes (one-half of the fetuses in each litter)
- Anogenital distance (AGD): Yes: (all viable fetuses)

Statistics:

Data Collected/Processed in ToxData:
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. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric:
- Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
- Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution were compared using the Mann Whitney test.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences.
Incidence:
- An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.
- No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss

Historical control data:

Historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source are avialable.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Piloerection was observed for five females treated at 200 mg/kg/day on various days starting on Day 9 or 10 post-coitum (Female Nos. 70, 73 and 82) or on Day 21 post-coitum only (Female Nos. 69 and 87). In addition, Female No. 70 was scored having a pale appearance on Days 9-15 post-coitum and Female No. 87 was noted with a hunched posture on Day 21 post-coitum. As these clinical signs were only observed in females treated at 200 mg/kg/day and these findings are in line with the clinical signs of animals that died preterm, these were considered to be test item-related.

Salivation seen after dosing was observed in all females treated at 100 mg/kg/day and in the majority of the females treated at 200 mg/kg/day, starting after multiple days of dosing. This was considered of no toxicological relevance, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.

Any other findings that were noted included alopecia and scabs. These findings 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 with the test item.

No clinical signs were noted in the control group and at 50 mg/kg/day.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

In total, 12 females did not survive until scheduled necropsy.
The death of eight females was considered test item-related; all these females were treated at 200 mg/kg/day.
Five females treated at 200 mg/kg/day were euthanized for animal welfare reasons during the treatment period. For further details please refer to 'Details on results'.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg/day, mean body weights were below the control mean during the whole study period, however only reaching statistical significance on Day 9 post-coitum (0.92x of control). Body weight gain was also lower on Day 9 post-coitum (mean body weight gain of 0% at 200 mg/kg/day vs. 4% in the concurrent control). For two females, body weight loss was noted between Day 18 and Day 21 post-coitum (Nos. 69 and 82). Overall, in females surviving until scheduled necropsy, body weight gain appeared to recover from Day 12 post-coitum onwards. At the end of the treatment period, body weights and corrected body weight gain were considered to be similar between the high dose group and concurrent control.

At 100 mg/kg/day, mean body weights were statistically significantly lower compared to control (up to 0.93x of control) on Days 6-12 post-coitum. However, mean body weight gain was comparable to the control group. This difference was most likely caused by the slightly lower mean body weight observed at 100 mg/kg/day compared to concurrent control on Day 2 post-coitum and was therefore considered unrelated to treatment with the test item.

At 50 mg/kg/day, no effects on body weight or body weight gain were observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg/day, lower mean (relative) food consumption was observed compared to control females on Days 6-9 and 18-21 post-coitum (-20% and -24% of control, respectively). For the remaining period, mean food consumption was comparable to control.

At 50 and 100 mg/kg/day, lower mean absolute food consumption was observed on Days 2-6 post-coitum (prior to initiation of dosing, and therefore not test item-related; -9% and -13% of control, respectively) compared to concurrent control. Normal food consumption was observed from Day 6-9 post-coitum onwards.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg/day, mean serum levels of thyroid stimulating hormone (TSH) were statistically significantly higher compared to concurrent control (2.2x of control). Although mean TSH concentration remained within the available historical control data, the individual serum levels of 5/12^3 females were above the 95-percentile (P95) of the historical control data (all individual concurrent control data were within the historical control data).
At 100 mg/kg/day, slightly increased mean serum levels of TSH (1.3x of control) was also recorded, however without reaching statistical significance and mean values within historical control data. All individual data, except of Female No. 65 (0.887 mU/L), were within available historical control data. However, as this increase was observed in the mid- and high-dose groups and based on the magnitude of change, this might be related to treatment with the test item.

Mean serum levels of triiodothyronine (T3) were statistically significantly lower at 200 mg/kg/day (0.65x of control). Although mean T3 concentration remained within the available historical control data2, the individual serum levels of 4/12 females were below the 5-percentile (P5) of this historical control data (all individual concurrent control data were within the historical control data). Based on the magnitude of change, a relation to treatment with the test item could not be excluded.

Mean serum levels of total thyroxine (Total T4) were slightly lower (0.9x of control) in all treated groups when compared to concurrent control, however, without reaching statistical significance. In the absence of a dose-related response, this was considered to be unrelated to treatment with the test item.

Endocrine findings:

not specified

Urinalysis findings:

not examined

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):

Test item-related significantly higher liver weights (absolute and/or relative to body weights) were noted in females as shown in the corresponding table under 'Any other information'. At 200 mg/kg/day, the absolute liver weight was increased by 23% and the relative liver weight by 30% compared to the control group.

There were no test item-related alterations in thyroid gland weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related macroscopic findings were present in the liver as:
- Focus/foci in 1/21 females treated at 50 mg/kg/day, in 1/22 females at 100 mg/kg/day and in 7/13 females at 200 mg/kg/day.
- Enlarged in 1/22 females treated at 100 mg/kg/day and in 6/13 females at 200 mg/kg/day.
- Irregular surface in 4/13 females treated at 200 mg/kg/day.

There were no test item-related gross thyroid gland observations. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic thyroid gland observations.

All of the recorded microscopic findings in the thyroid gland were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

Mortality:
Female Nos. 74, 76, 79 and 81 were sacrificed in extremis on Day 9 post coitum based on body weight loss (between -9 and -12% compared to Day 6 post-coitum). Food consumption observed on Days 6-9 post-coitum was severely reduced in these females. For Female No. 76, clinical signs included hunched posture, piloerection and pale appearance on the day prior to necropsy. No clinical signs were observed for the other three females. At necropsy it was noted that the liver of all four females was pale, enlarged, hardened and/or had several dark red foci. In addition, the thymus of Female No. 76 was reduced in size, and Female No. 81 had isolated dark red foci on the outside of the stomach wall and ectopic splenic tissue.

Female No. 85 was sacrificed in extremis on Day 20 post-coitum based on clinical signs observed on this day. These clinical signs included lethargy, hunched posture, labored respiration and piloerection. Salivation was observed from Days 8-19 post-coitum. Normal body weight gain and food consumption was observed prior its sacrifice. Necropsy revealed a hardened liver with an irregular surface with many tan-colored foci.

Three females treated at 200 mg/kg/day were found dead on Day 20 or 21 post-coitum: Female Nos. 77 and 80 were found dead on the day of scheduled necropsy (Day 21 post-coitum). Both females were observed with normal body weight gain and food consumption up to Day 18 post-coitum. Subsequently, these females showed body weight loss (-8% and -5% compared to Day 18 post-coitum, respectively) and food consumption below normal values on Day 21 post-coitum. For Female No. 77, piloerection was observed on Days 10-14 and 21 post-coitum. Relevant clinical signs of Female No. 80 included hunched posture on Days 8-13 post-coitum. Both females showed autolysis at necropsy. Macroscopic findings included an irregular liver surface with many tan-colored foci (both females) and a hardened liver (Female No. 80 only).

Female No. 84 was found dead on Day 20 post-coitum. Normal body weight gain and food consumption was observed up to and including Day 15 post-coitum. However, a reduction in body weight of 11% compared to Day 15 post-coitum was observed on Day 18 post-coitum. In addition, food consumption on Day 15-18 post-coitum was below normal values. Clinical signs prior to its death included piloerection on Days 17-19 post-coitum and salivation from Day 8 post-coitum onwards. During necropsy, this female was observed being autolytic, which can be explained by the fact that this animal was already dead before necropsy. Other macroscopic findings included an irregular liver surface with many tan colored foci.
The females sacrificed for animal welfare reasons or that were found dead were all pregnant at the time of necropsy.

Three females were euthanized as they started to deliver their litters: Female No. 1 (control) and Female No. 23 (50 mg/kg/day) started to deliver on the day of scheduled necropsy (Day 21 post-coitum). Female No. 5 (control) was euthanized on Day 20 post-coitum, after it started to deliver its litter early. All females were observed with a normal body weight (gain) and food consumption prior to Day 21 post-coitum. In addition, no clinical signs or macroscopic findings were observed. Early deliveries are occasionally seen in this type of study. Given the absence of a dose-related trend and as it occurred in two control females, this was considered unrelated to treatment with the test item.

Female No. 5 (control) started to deliver its litter on Day 20 post-coitum and was therefore euthanized preterm. As a result, body weight (gain) and food consumption were not determined on Day 21 post-coitum, and corrected body weight gain and organs weights were not collected. Normal body weight (gain) and food consumption prior to Day 21 post-coitum were observed and no clinical signs or macroscopic findings were present.

In addition, Female No. 83 (200 mg/kg/day) was found dead on Day 9 post-coitum. Other than piloerection observed on Day 8 post-coitum, no clinical signs were observed prior to its death. At necropsy, this female had beginning autolysis. In addition, foamy contents in the trachea, the lungs were not collapsed and contained black foci and dark red foci on the liver were observed. Based on the lung findings, the death of this female was considered to be related to the gavage procedure. Therefore, this death was considered unrelated to treatment with the test item.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

At scheduled necropsy, all females were pregnant, except for Female No. 2 (control group). Female No. 24 (50 mg/kg/day) was gravid but was observed with resorptions only. The females sacrificed for animal welfare reasons preterm or found dead (see information given under 'Mortality') were all pregnant at the time of necropsy.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The number of corpora lutea and implantation sites, and pre-implantation loss in the control and test groups up to 100 mg/kg/day were comparable and in the range of normal biological variation.

Total litter losses by resorption:

not specified

Early or late resorptions:

no effects observed

Description (incidence and severity):

Early or late resorptions were considered not affected by treatment up to 100 mg/kg/day.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

Viable or dead fetuses were considered not affected by treatment up to 100 mg/kg/day.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

not examined

Details on maternal toxic effects:

Maternal toxicity: yes. Treatment with diallyl 2,2’-oxydiethyl dicarbonate resulted in a significant level of maternal toxicity at 200 mg/kg/day.

In the present study, the number of females with viable litters for evaluation was 19, 20, 22 and 12 in the control, 50, 100 and 200 mg/kg/day groups, respectively. As according to the guidelines, a minimum of 16 litters is required for evaluation of developmental data, the 12 litters available at 200 mg/kg/day were not sufficient for a robust and valid evaluation of developmental data. As such, developmental data of Group 4 were excluded from reporting (but kept in the raw data), to avoid bias and misinterpretation as the data at 200 mg/kg/day is incomplete.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

mortality

2

Dose descriptor:

NOAEL

Remarks:

maternal developmental toxicity

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: Overall maternal developmental effects

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no test item-related effects on fetal body weights (both sexes) noted by treatment with the test item up to 100 mg/kg/day.

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment with the test item up to 100 mg/kg/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no test item-related effects on litter size noted after treatment with the test item up to 100 mg/kg/day.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

There were no test item-related effects on fetal anogenital distance (both sexes) noted after treatment with the test item up to 100 mg/kg/day.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No external malformations and external variations observed following treatment up to 100 mg/kg/day.

Skeletal malformations:

no effects observed

Description (incidence and severity):

There were no test item-related effects on skeletal morphology following treatment up to 100 mg/kg day.

In the control group, two fetuses were observed with skeletal malformations. Fetus No. A014-06 was observed with bent limb bones and Fetus No. A015-03 was observed with a vertebral centra anomaly. As both malformations only occurred in fetuses of the control group, these malformations were as such considered to be of spontaneous origin.

All skeletal variations occurred in the absence of a dose-related incidence trend, infrequently, in control fetuses only and/or at frequencies that were within the range of available historical control data. Therefore, they were considered to be unrelated to treatment with the test item.

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no test item-related effects on visceral morphology following treatment up to 100 mg/kg/day.

At 50 mg/kg/day, two fetuses (Nos. A025-02 and A028-03) were observed with the malformation “small eye”, which was observed at soft tissue cephalic examination. Due to the occurrence at the low dose and as the % per litter remained within the maximum of the historical control data, the occurrence of this malformation was considered to be a chance finding.

The three visceral variations that were noted (small supernumerary liver lobes, dilated ureter and absent renal papilla) occurred at low incidences and in the control and/or 50 mg/kg/day groups only. Therefore, they were considered not related to treatment with the test item.

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

Embryoticity/ teratognic effects: No.
Up to 100 mg/kg/day, no test item-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, anogenital distance, external, visceral and skeletal malformations and developmental variations).

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall embryotoxic / teratogenic effects

Remarks on result:

other: Absence of any test item-related effects on fetal observations

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Results

Dose Formulation Analyses:

- Accuracy: The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulations.

- Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Organ Weights:

Dose level (mg/kg/day):	50	100	200
LIVER
Absolute	4	5	23**
Relative to body weight	6	9*	30**

*: P<0.05, **: P<0.01

Fetal Findings: In the present study, the number of females with viable litters for evaluation was 19, 20, 22 and 12 in the control, 50, 100 and 200 mg/kg/day groups, respectively. As according to the guidelines, a minimum of 16 litters is required for evaluation of developmental data, the 12 litters available at 200 mg/kg/day were not sufficient for a robust and valid evaluation of developmental data. As such, developmental data of Group 4 were excluded from reporting (but kept in the raw data), to avoid bias and misinterpretation as the data at 200 mg/kg/day is incomplete.

Applicant's summary and conclusion

Conclusions:

Based on the results of this prenatal developmental toxicity study (i.e. adverse effects on body weight and food consumption and unscheduled deaths in dams treated at 200 mg/kg/day) the maternal No Observed Adverse Effect Level (NOAEL) for diallyl 2,2’-oxydiethyl dicarbonate was established as being 100 mg/kg/day.
Due to the absence of any test item-related effects on fetal observations, the developmental No Observed Adverse Effect Level (NOAEL) for diallyl 2,2’-oxydiethyl dicarbonate was established as being at least 100 mg/kg/day.

Note: In the present study, the number of females with viable litters for evaluation was 19, 20, 22 and 12 in the control, 50, 100 and 200 mg/kg/day groups, respectively, after exclusion of non-pregnant animals, animals showing an early delivery, and animals that did not survive until scheduled necropsy. As according to the guidelines, a minimum of 16 litters is required for evaluation of developmental data, the 12 litters available at 200 mg/kg/day were not sufficient for a robust and valid evaluation of developmental data. As such, developmental data of Group 4 were excluded from reporting, to avoid bias and misinterpretation as this data is incomplete.

Executive summary:

The present study was conducted according to OECD 414 (adopted June 2018) and in compliance with GLP. In the study time-mated female Wistar Han rats were treated with diallyl 2,2’-oxydiethyl dicarbonate from Day 6 to 20 post-coitum, inclusive by daily oral gavage at dose levels of 50, 100 and 200 mg/kg/day. The rats of the control group received the vehicle, polyethylene glycol 400, alone.

The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), gross necropsy findings, organ weights (liver and thyroid gland), uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and post-implantation loss. In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations.

Maternal toxicity

Treatment with diallyl 2,2’-oxydiethyl dicarbonate resulted in a significant level of maternal toxicity at 200 mg/kg/day. Test item-related mortality was observed at 200 mg/kg/day, consisting of five females that were euthanized for animal welfare reasons on Day 9 or 20 post-coitum and three females found dead on Day 20 or 21 post-coitum. All females euthanized in extremis were observed with body weight loss and food consumption below normal values and/or clinical signs including lethargy, hunched posture, labored respiration, piloerection and/or pale appearance.

Females that were found dead were observed with body weight loss and food consumption below normal values during the period adjacent to their death but with normal body weight gain and food consumption in the period before that. Clinical signs included piloerection or hunched posture. At necropsy, it was observed that all females had liver findings, including pale appearance of the liver, enlarged and/or hardened liver, dark red or tan colored foci on the liver and/or an irregular surface of the liver. In addition, isolated dark red foci on the outside of the stomach wall or a thymus that was reduced in size were observed in two of the females.

Piloerection was observed for five females treated at 200 mg/kg/day on various days starting on Day 9 or 10 post-coitum or was present on Day 21 post-coitum only. In addition, one female had a pale appearance on Days 9-15 post-coitum and another female showed a hunched posture on Day 21 post-coitum. As these clinical signs were only observed in females treated at 200 mg/kg/day and these findings are in line with the clinical signs of animals that died preterm, these effects were considered as an indication for an adverse test item-related effect.

Overall, mean food consumption was lower in females at 200 mg/kg/day on Days 6-9 and Days 18-21 post-coitum compared to control. For the remaining period, mean food consumption was comparable to control. In addition, mean body weight gain was statistically significantly lower on Day 9 post-coitum compared to control (which was most likely caused by the four females that were sacrificed for animal welfare reasons on Day 9 post-coitum).

For two females, body weight loss was noted between Day 18 and Day 21 post-coitum, indicating a similar pattern as the moribund animals. Despite the apparent recovery, given the size of the observed effects on body weight gain and food consumption resulting in sacrifice or death of eight high-dose females, these effects were considered adverse.

Macroscopic observation at necropsy revealed foci on the liver (statistically significant) as well as enlargement and/or hardening of the liver and an irregular surface of the liver for the majority of the remaining females at 200 mg/kg/day. These findings were also found in the females that were prematurely sacrificed due to body weight loss and food consumption below normal values and/or in the females that were found dead. In addition, liver weights (both absolute and relative to body weight) were also statistically significantly higher compared to concurrent control. Although histopathological evaluation was not performed, given the severity of these macroscopic findings they were considered adverse. Based on the low incidence of liver findings and the small magnitude of change in liver weights at 50 and 100 mg/kg/day, and in the absence of detrimental effects on animal welfare at these dose levels, the effects on the liver (presence of macroscopic findings and higher liver weights) were considered to be non-adverse. Higher mean serum levels of thyroid stimulating hormone (TSH) were observed at 100 and 200 mg/kg/day compared to concurrent control, reaching statistical significance at 200 mg/kg/day only. However, mean TSH concentrations remained within the available historical control data. Lower mean serum levels of triiodothyronine (T3) were observed in all treated groups compared to concurrent control, only reaching statistical significance at 200 mg/kg/day. However, mean T3 concentration remained within the available historical control data. No test item-related significant changes were noted in any of the remaining maternal parameters investigated in this study (i.e. thyroxine (T4) and histopathologic examination of the thyroid gland). Excluding non-pregnant females, females that started to deliver their litter and females that did not survive until scheduled necropsy, there were 19 females in the control group, 20 females at 50 mg/kg/day, 22 females at 100 mg/kg/day and 12 females at 200 mg/kg/day with viable litters available for developmental evaluation. As according to the guidelines, a minimum of 16 litters is required for evaluation of developmental data, the 12 litters available at 200 mg/kg/day were not sufficient for a robust and valid evaluation of developmental data. As such, developmental data of Group 4 were excluded from reporting (but kept in the raw data), to avoid bias and misinterpretation as the data at 200 mg/kg/day is incomplete. The number of corpora lutea, implantation sites, viable or dead fetuses, early or late resorptions, and pre- and post-implantation loss were considered not affected by treatment up to 100 mg/kg/day.

 

Developmental toxicity

Up to 100 mg/kg/day, no test item-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, anogenital distance, external, visceral and skeletal malformations and developmental variations).

In conclusion, based on the results of this prenatal developmental toxicity study (i.e. adverse effects on body weight and food consumption and unscheduled deaths in dams treated at 200 mg/kg/day) the maternal No Observed Adverse Effect Level (NOAEL) for diallyl 2,2’-oxydiethyl dicarbonate was established as being 100 mg/kg/day. Note: In the present study, according to the OECD 414 guidelines, an insufficient number of litters was available for evaluation at 200 mg/kg/day. As such, developmental data of Group 4 were excluded from reporting, to avoid bias and misinterpretation as this data is incomplete.

Due to the absence of any test item-related effects on fetal observations, the developmental No Observed Adverse Effect Level (NOAEL) for diallyl 2,2’-oxydiethyl dicarbonate was established as being at least 100 mg/kg/day.