Diallyl 2,2'-oxydiethyl dicarbonate

Endpoint summary

Description of key information

Key, rat, oral, OECD 408, GLP, NOAEL = 25 mg/kg bw/d

Key, rat, dermal, OECD 422, GLP, NOAEL > 1030 mg/kg bw/d

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

A GLP-compliant study according to OECD Guideline 408 (Klimisch 1) is available. Therefore, the quality of the database is high.

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 12, 2003-October 20, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well documented GLP guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan
- Age at study initiation: eight weeks (six at arrival plus two-week acclimation period)
- Weight at study initiation: All animals placed on study had body weights within ±20% of the mean body weight for each sex. Forty male rats (weighing 223 to 292 g at randomization) and 80 female rats (weighing 163 to 205 g at randomization)
- Fasting period before study: No, except overnight prior to blood sample collection.
- Housing: Animals were individually housed in stainless steel cages with wire mesh fronts and bottoms, suspended over pans containing absorbent liners.
- Diet (e.g. ad libitum): yes, except during designated fasting periods. Diet (meal Lab Diet® Certified Rodent Diet® #5002, PMI Nutrition International, Inc.)
- Water (e.g. ad libitum): Tap water was available ad libitum to all animals and supplied via an automatic watering system.
- Acclimation period: two weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 67 to 74°F [monitored and recorded daily]
- Humidity (%): 21 to 69% [monitored and recorded daily]
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was provided for approximately 12 hours per day via an automatic timer.

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: Test areas: an anterior and posterior site on the back. The dose sites were alternated daily between the anterior and posterior areas.
- % coverage: no less than 10% of the total body surface.
- Type of wrap if used: The control and test article were drawn into a plastic 1 mL syringe (except for the 150 mg/kg/day groups, which required a 100 µL glass syringe), administered from the hub of the syringe, and distributed evenly over the appropriate site.
- Time intervals for shavings or clipplings: at weekly intervals throughout the study, all animals had hair clipped from the test areas.


REMOVAL OF TEST SUBSTANCE
- Washing (if done): the sites were washed with warm soapy water, and rinsed with tepid tap water.
- Time after start of exposure: six hours after test article administration.


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): at dose levels of 150, 454 and 1030 mg/kg/day at respective dose volumes of 0.13, 0.4, and 0.9 mL/kg.
- Constant volume or concentration used: yes. The dose volumes were derived on the basis of the density of the test article, 1.143 g/mL.


CONTROL MATERIAL

- Amount(s) applied (volume or weight with unit): based on the most recent body weightt at dose volume of 0.9 mg/mL
- Concentration (if solution): not applicable
- Lot/batch no. (if required): 03-144-JT-03, 04-146-JT, 04-100-JT
- Purity: Documentation on the strength, purity, composition, stability, physical properties, and other pertinent information on each batch of control article (0.9% Sodium Chloride for Injection, USP) was limited to that information listed on the label of this commercially available control article.


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes. Immediately following dosing, the application site was covered with gauze dressing and secured with nonirritating tape. Six hours after test article administration, the gauze and tape were removed, the sites washed and rinsed. The dose site of each animal was blotted dry prior to returning to the cage.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A compositional analysis was conducted on test article samples (2 g/sample) collected prior to initiation of dosing (start of study) and after completion of test article administration (end of study). The analysis was performed to confirm the identity of the test article by HPLC TAN and NMR procedures.All compositional analytical work was conducted by Ricerca, LLC, Concord, Ohio. The work performed by Ricerca in conjunction with this study was conducted in compliance with GLP regulations and subjected to review by the Quality Assurance Unit (QAU) of that laboratory.

Duration of treatment / exposure:

The test and control articles were administered for at least 42 consecutive days to males (14 days premating, 14 days of mating, and 14 days after completion of the mating period), at least 42 days to females.

Frequency of treatment:

once per day at approximately the same time each day

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

454 mg/kg bw/day (nominal)

Dose / conc.:

1 030 mg/kg bw/day (nominal)

No. of animals per sex per dose:

- ten male and ten female at dose levels of 150, 454 and 1030 mg/kg bw/day in repeat dose component
- ten female at dose levels of 150, 454 and 1030 mg/kg bw/day in reproductive component

Control animals:

other: concurrent with saline

Details on study design:

- Dose selection rationale: The dose levels were selected in consultation with the Sponsor, on the basis of available data from previous studies.
- Rationale for animal assignment (if not random): randomized
- Rationale for selecting satellite groups: not applicable
Control groups, treated only with vehicle alone, were needed to exclude any effect of vehicle and make possible to interpret the effects of test material
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): randomized

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: twice daily (once during test or control article administration, and once following completion of the six-hour exposure and removal of the wrap) for morbidity, mortality, signs of injury, and the availability of food and water.
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the start of treatment and weekly during the study.
Deatiled clinical observations checked in table [No.1] were included

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily for the first 20 days and weekly for the remainder of the study for erythema and edema.
The evaluations were made according to a skin reaction scale based on the Draize1 scale for scoring skin irritation. Evaluations were conducted prior to test or control article administration that day. The daily observations occurring between weekly intervals are not reported, but are maintained in the study data.
Dermal irritation checked in table [No.1] were included

BODY WEIGHT AND BODY WEIGHT GAIN: Yes
- Time schedule for examinations: the day after arrival, at randomization, at initiation of test or control article administration, weekly during the study, and at termination.


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, but in g food/animal/day
Individual food consumption for males was measured and recorded weekly during the study, except during the two-week mating period. Individual food consumption for repeat dose females was measured and recorded weekly during the study.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND COAGULATION: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes, carbon dioxide/oxygen
- Animals fasted: Yes, overnight prior to sample collection, but had free access to drinking water
- How many animals: all males and repeat dose females
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:at termination from all males and repeat dose females
- Animals fasted: Yes, overnight prior to sample collection, but had free access to drinking water
- How many animals: from all males and repeat dose females
- Parameters checked in table [No.1] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to initiation of test or control article administration, at the start of treatment, and weekly during the study.
- Dose groups that were examined: each repeat dose male and female
- Battery of functions tested in table [No.1] were included.

BEHAVIORAL OBSERVATION-REPEAT DOSE COMPOMENT:

- Time schedule for examinations: prior to the first exposure and during the last week of study.
- Dose groups that were examined: All males and repeat dose females
- Battery of functions tested in table [No.1] were included.

OTHER:
Fo BREEDING PROCEDURES- REPRODUCTIVE COMPONENT (see in section 7.8 Toxicity to reproduction)
Fo PARTURITION AND Fi LITTER OBSERVATIONS - REPRODUCTIVE COMPONENT (see in section 7.8 Toxicity to reproduction)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (table 2), all adult animals dying spontaneously or euthanized at scheduled necropsies were externally examined and any abnormalities were identified and correlated with antemortem findings. The carcasses were discarded without further evaluation.
Organs and tissues from all adult animals were removed, trimmed of any adherent tissues, weighed (where required) and placed in neutral buffered formalin, except for the testes and epididymides which were placed in Bouin's fixative, and the eyes which were placed in Davidson's fixative. The lungs were infused via the trachea with formalin. The urinary bladder was inflated with formalin at necropsy and examined internally after fixation.

HISTOPATHOLOGY: Yes, were performed for all males and repeat dose females in the control and high dose groups.
Microscopic examination of formalin-fixed hematoxylin and eosin-stained paraffin sections of the tissues were performed for all males and repeat dose females in the control and high dose groups. For processing of the testes, hematoxylin and period acid Schiff (PAS) staining and paraffin embedding was performed.

NEUROPATHOLOGICAL EVALUATIONS
Nervous system tissue (cervical, thoracic, and lumbar spinal cord) from female animals from the mid and high repeat dose groups (454 and 1030 mg/kg bw/day, respectively) as well as the control were sent to Experimental Pathology Laboratories, Inc. (EPL*), in Henidon, Virgina, for further processing and/or special neuropathology staining which allowed for better examination of possible nervous system effects. The sciatic nerve, when available, was processed to glycomethacrylate blocks and then sectioned and stained with Toluidine Blue or Bielschowsky for histopathologic evaluation.

Other examinations:

ORGAN WEIGHTS
Body and protocol-specified organ weights were recorded at necropsy and appropriate organ weight ratios were calculated (absolute and relative to body and brain weight) for all adult animals. Paired organs were weighed together. Organs were not weighed for animals dying spontaneously.

Statistics:

Group Pair-wise Comparisons
For each specified endpoint and for all collection intervals, Levene's test 5 was used to assess homogeneity of group variances. If Levene's test was not significant (p>0.01), Dunnett's test6 was used to compare each treatment group with the control group. If Levene's test was significant (p<0.01), comparisons with the control group were made using Welch's t-test7 with a Bonferroni correction. Results of all pair-wise comparisons were reported at the 0.05 and 0.01 significance levels. All endpoints were analyzed using two-tailed tests. Besides these tests, Log Transformation, Fisher's Exact Test, Covariate Analysis and Arcsin-Square-Root Transformation were used

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Mortality:

no mortality observed

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

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
All treated animals in the repeat dose component survived to scheduled euthanasia.
No effect of treatment with ADC was evident from the general and detailed clinical examinations of the repeat dose animals. The few findings seen among the treated animals occurred with low incidence or with similar frequency as controls. These findings (i.e. red material around the eyes or nose, sparse amounts of hair on the fore- and hindlimbs/paws) are seen commonly in this laboratory with this strain and age of animal and the occurrences in this study were considered unrelated to treatment.

BODY WEIGHT AND WEIGHT GAIN
No effect of treatment with ADC was evident from body weight data for males and females in the repeat dose component. Mean body weights for the treated animals were comparable to controls throughout the seven-week treatment study.
No effect of treatment with ADC was evident from body weight gain for males and females in the repeat dose component. Mean week-to-week body weight gain for the treated animals was comparable to controls. In the females there was a mean weight loss during the last week of study. This was seen in all groups (control and treated) and was attributed to half of the females in each group being fasted for blood collection (clinical pathology) overnight prior to euthanasia and necropsy. The remaining females in each group were necropsied the following day and were not fasted overnight.
FOOD CONSUMPTION
No effect of treatment with ADC was evident from food consumption for the males and females in the repeat dose component. Mean food consumption for the males during the two weeks prior to pairing and two weeks following completion of the pairing (mating) period was comparable to controls. Likewise, food consumption for the treated females in the repeat dose component over the entire six-week treatment period was comparable to controls.

HAEMATOLOGY
There were no adverse test article-related effects on hematology parameters evaluated. The red cell indices (MCHC, MCV, and MCH) were statistically decreased in males at 454 mg/kg/day. The differences in MCHC, MCV, and MCH were minimal, however, and not considered to be treatment related.

CLINICAL CHEMISTRY
No adverse effects were seen in clinical chemistry analytes. Few analytes in the treated groups exhibited statistically significant differences from controls, but none were considered physiologically or toxicologically relevant.

NEUROBEHAVIOUR
No effect of treatment with ADC was evident from the neurobehavioral evaluations of the repeat dose animals.

BEHAIVIOUR
Auditory Response, Corneal Reflex, Pupil Response, and Grip Strength

No effect of treatment with ADC was evident from auditory response, corneal reflex or pupil response. With the exception of one control female which did not pass the corneal reflex evaluation, all other animals (control and treated groups) passed these behavioral tests at terminal evaluation. Similarly, no effect of treatment was evident in either males or females from forelimb grip strength evaluations. Forelimb grip strength in the 150 mg/kg/day males was statistically lower than controls; however, in the absence of a similar response in males at the higher dose levels, this was considered spurious and unrelated to treatment. In both sexes, forelimb grip strength at termination was greater than at the pretest evaluation.

No effect of treatment with ADC to males was evident from hindlimb grip strength. These values in the treated males at terminal examination were comparable to controls. Grip strength in the control and treated males at terminal examination was notably lower than at pretest and differences ranged from about 43% lower in controls to 23% lower in the 1030 mg/kg/day group. During this pretest interval, hindlimb grip strength in the 150 and 1030 mg/kg/day males was statistically lower than controls and comparable to controls in the 454 mg/kg/day group.

Mean hindlimb grip strength at terminal examination among females treated at 454 and 1030 mg/kg/day was statistically significantly lower than the control group; both the mid-and high-dose groups displayed a very similar decrease. This finding appears to be spurious and not toxicologically significant because: 1) the effect was not dose responsive (grip strength was similar for the two dose groups despite the two-fold increase in dose between the mid- and high-dose groups), 2) mean hindlimb grip strength was significantly lower at termination than prior to the start of the study for all groups (treatment and control), 3) the effect was noted only in females and not in males, 4) there were no microscopic changes noted in H&E stained sections of the spinal cord or sciatic nerve or hindlimb muscle, and 5) further processing and/or special neuropathology staining of the spinal cord and sciatic nerve also did not reveal any microscopic changes that were believed to be treatment related.

Motor activity
Motor activity evaluated as horizontal activity, vertical activity, total distance, and stereotypy over a 20-minute trial was comparable to controls in the treated females and in the 150 mg/kg/day males at the terminal evaluation. Motor activity was higher than control in the 454 and 1030 mg/kg/day males during the 5-10 minute testing interval, and in the 454 mg/kg/day males over the entire 0-20 minutes of testing and in most instances these differences were statistically significant. For all other intervals (0-5, 10-15, and 15-20 minutes) motor activity for these treated males did not differ statistically from controls and was considered comparable between the groups. The increased activity in these treated males during this one five minute interval over the entire 20-minute period was not considered toxicologically meaningful since there was no clear dose response in any of the activity parameters. Mean values for each activity (horizontal, vertical, total distance, and stereotypy) were similar between the 454 and 1030 mg/kg/day groups. Likewise, cumulative evaluations over the entire 20-minute testing period were also not dose responsive, and only differed statistically from control at the 454 mg/kg/day dose level. During the pretest evaluation, motor activity for the treated males and females (all groups) was comparable to controls.
Emotionality
No effect of treatment with ADC was evident from the emotionality evaluations conducted during the first five minutes of the motor activity testing. The parameters recorded during these evaluations (intervals of urination and counts of defecation, grooming, backing and rearing) for the treated groups were comparable to controls.

ORGAN WEIGHTS
No test article-related organ weight changes were noted in rats of either sex that received ADC in the repeat dose phase of the study.
Absolute mean prostate weights and relative prostate weights to body and brain weights were statistically significantly increased in male rats that received 454 mg/kg/day and 1030 mg/kg/day ADC compared to controls. However, there were no corresponding microscopic changes in prostates of males in these groups. The prostate weight increase was not considered to be toxicologically significant. Absolute mean heart weight and relative heart weight to body and brain weights were statistically significantly increased in female rats that received 1030 mg/kg/day ADC compared to controls, but males were not affected and there were no corresponding microscopic changes in hearts. The heart weight increase was not considered to be toxicologically significant.

GROSS PATHOLOGY
No test article-related macroscopic changes were observed in rats of either sex.

HISTOPATHOLOGY: NON-NEOPLASTIC
No test article-related microscopic changes were noted in rats of either sex that received repeated doses of ADC. Microscopic changes observed were typical of those commonly found in animals of the same strain and age and were considered to be incidental.

OTHER FINDINGS
NEOROPATHOLOGICAL EVALUATIONS
The purpose of these additional neuropathology evaluations was to determine if the decrease in hindlimb grip strength in mid-and high dose females was associated with any neuropathological findings. Cervical, thoracic and lumbar spinal cord tissues were submitted for histopathological evaluation
In the neuropathological evaluations of selected nervous tissues conducted by EPLS (Report No: 139-019), minimal axonal degeneration characterized by either the presence of digestion chambers and /or swollen axons (spheroids) was observed in one level of spinal cord from one high-dose (1030 mg/kg/day) female and from one mid-dose (454 mg/kg/day) female. The sciatic nerve of one high-dose female and two mid-dose females also had minimal axonal degeneration. Similar minimal changes were not observed in the control animals. These minimal lesions were considered well within the range of what could occur as an incidental background finding. Thus, it was concluded that since there was no dose response, the changes seen were probably incidental background findings unrelated to test article treatment.

Dose descriptor:

NOAEL

Effect level:

>= 1 030 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: There were no toxicologically significant treatment related effects in all parameters tested

Critical effects observed:

not specified

Conclusions:

In this rat dermal repeat dose toxicity study with a reproduction/developmental toxicity component, the No-Observable-Adverse-Effect Level (NOAEL) of the test article diallyl diglycol carbonate, for parental toxicity was 1030 mg/kg/day, the highest dose level evaluated.

Executive summary:

This study was conducted in accordance with OECD Guideline 422, which is comprised of two components, a repeat dose toxicity study with neurobehavioral evaluations and a reproduction/developmental toxicity screening study (see section 7.8 Toxicity to reproduction for summary).

The purpose of the repeat dose toxicity component was to provide information on possible target organ effects and the potential for neurobehavorial effects arising from repeated exposures. Each repeat dose group contained ten male and ten female Sprague-Dawley rats [Crl: CD® (SD)IGS BR]. The ten male animals of each repeat dose group were also utilized for the reproductive/developmental component of the study. Animals were administered dermally once daily via occlusion for 6 hours at dose levels 150, 454, and 1030 mg/kg/day for at least 42 consecutive days. The control animals received 0.9% Sodium Chloride, USP, at a volume of 0.9 mL/kg for the same duration as the treated animals. Observations of the animals included clinical signs, neurobehavioral observations (repeat dose component only), dermal evaluation, body weights, and food consumption. Evaluations for motor activity, emotionality, and other behavioral observations, including auditory response, grip strength, pupil reflex, and corneal reflex, were conducted for all repeat dose animals, pretest and prior to scheduled terminal euthanasia (after seven weeks of treatment). Blood collections for clinical pathology evaluations were conducted at study termination. Complete necropsies were performed on all animals (repeat dose and reproductive components) and organs and tissues were collected, weighed, and preserved. Organs and tissues from control and high-dose animals in the repeat dose component were examined microscopically. Nervous system tissues (cervical, thoracic, and lumbar spinal cord and sciatic nerve) from female animals from the mid and high dose groups (454 and 1030 mg/kg bw/day) as well as the control were evaluated for neuropathology.

No effect of treatment was evident from mortality, clinical evaluations, neurobehavioral evaluations, dermal evaluations, body weights, food consumption, hematological evaluations, serum clinical chemistry, organ weights, macroscopic, or microscopic evaluations. Decreased hindlimb grip strength as well as axonal dgenerations in spinal cord and sciatic nerve were noted at termination for female animals in the 454 and 1030 mg/kg/day dose groups (Table 2. in "Remarks on results"). These findings appear to be spurious and not toxicologically significant.

Thus, in this rat dermal repeat dose toxicity study with a reproduction/developmental toxicity component, the No-Observable-Adverse-Effect Level (NOAEL) of the test article diallyl diglycol carbonate, for parental toxicity was 1030 mg/kg/day, the highest dose level evaluated.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 030 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

A GLP-compliant study according to OECD Guideline 422 (Klimisch 1) is available. Therefore, the quality of the database is high.

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 12, 2003-October 20, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well documented GLP guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan
- Age at study initiation: eight weeks (six at arrival plus two-week acclimation period)
- Weight at study initiation: All animals placed on study had body weights within ±20% of the mean body weight for each sex. Forty male rats (weighing 223 to 292 g at randomization) and 80 female rats (weighing 163 to 205 g at randomization)
- Fasting period before study: No, except overnight prior to blood sample collection.
- Housing: Animals were individually housed in stainless steel cages with wire mesh fronts and bottoms, suspended over pans containing absorbent liners.
- Diet (e.g. ad libitum): yes, except during designated fasting periods. Diet (meal Lab Diet® Certified Rodent Diet® #5002, PMI Nutrition International, Inc.)
- Water (e.g. ad libitum): Tap water was available ad libitum to all animals and supplied via an automatic watering system.
- Acclimation period: two weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 67 to 74°F [monitored and recorded daily]
- Humidity (%): 21 to 69% [monitored and recorded daily]
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was provided for approximately 12 hours per day via an automatic timer.

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: Test areas: an anterior and posterior site on the back. The dose sites were alternated daily between the anterior and posterior areas.
- % coverage: no less than 10% of the total body surface.
- Type of wrap if used: The control and test article were drawn into a plastic 1 mL syringe (except for the 150 mg/kg/day groups, which required a 100 µL glass syringe), administered from the hub of the syringe, and distributed evenly over the appropriate site.
- Time intervals for shavings or clipplings: at weekly intervals throughout the study, all animals had hair clipped from the test areas.


REMOVAL OF TEST SUBSTANCE
- Washing (if done): the sites were washed with warm soapy water, and rinsed with tepid tap water.
- Time after start of exposure: six hours after test article administration.


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): at dose levels of 150, 454 and 1030 mg/kg/day at respective dose volumes of 0.13, 0.4, and 0.9 mL/kg.
- Constant volume or concentration used: yes. The dose volumes were derived on the basis of the density of the test article, 1.143 g/mL.


CONTROL MATERIAL

- Amount(s) applied (volume or weight with unit): based on the most recent body weightt at dose volume of 0.9 mg/mL
- Concentration (if solution): not applicable
- Lot/batch no. (if required): 03-144-JT-03, 04-146-JT, 04-100-JT
- Purity: Documentation on the strength, purity, composition, stability, physical properties, and other pertinent information on each batch of control article (0.9% Sodium Chloride for Injection, USP) was limited to that information listed on the label of this commercially available control article.


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes. Immediately following dosing, the application site was covered with gauze dressing and secured with nonirritating tape. Six hours after test article administration, the gauze and tape were removed, the sites washed and rinsed. The dose site of each animal was blotted dry prior to returning to the cage.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A compositional analysis was conducted on test article samples (2 g/sample) collected prior to initiation of dosing (start of study) and after completion of test article administration (end of study). The analysis was performed to confirm the identity of the test article by HPLC TAN and NMR procedures.All compositional analytical work was conducted by Ricerca, LLC, Concord, Ohio. The work performed by Ricerca in conjunction with this study was conducted in compliance with GLP regulations and subjected to review by the Quality Assurance Unit (QAU) of that laboratory.

Duration of treatment / exposure:

The test and control articles were administered for at least 42 consecutive days to males (14 days premating, 14 days of mating, and 14 days after completion of the mating period), at least 42 days to females.

Frequency of treatment:

once per day at approximately the same time each day

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

454 mg/kg bw/day (nominal)

Dose / conc.:

1 030 mg/kg bw/day (nominal)

No. of animals per sex per dose:

- ten male and ten female at dose levels of 150, 454 and 1030 mg/kg bw/day in repeat dose component
- ten female at dose levels of 150, 454 and 1030 mg/kg bw/day in reproductive component

Control animals:

other: concurrent with saline

Details on study design:

- Dose selection rationale: The dose levels were selected in consultation with the Sponsor, on the basis of available data from previous studies.
- Rationale for animal assignment (if not random): randomized
- Rationale for selecting satellite groups: not applicable
Control groups, treated only with vehicle alone, were needed to exclude any effect of vehicle and make possible to interpret the effects of test material
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): randomized

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: twice daily (once during test or control article administration, and once following completion of the six-hour exposure and removal of the wrap) for morbidity, mortality, signs of injury, and the availability of food and water.
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the start of treatment and weekly during the study.
Deatiled clinical observations checked in table [No.1] were included

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily for the first 20 days and weekly for the remainder of the study for erythema and edema.
The evaluations were made according to a skin reaction scale based on the Draize1 scale for scoring skin irritation. Evaluations were conducted prior to test or control article administration that day. The daily observations occurring between weekly intervals are not reported, but are maintained in the study data.
Dermal irritation checked in table [No.1] were included

BODY WEIGHT AND BODY WEIGHT GAIN: Yes
- Time schedule for examinations: the day after arrival, at randomization, at initiation of test or control article administration, weekly during the study, and at termination.


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, but in g food/animal/day
Individual food consumption for males was measured and recorded weekly during the study, except during the two-week mating period. Individual food consumption for repeat dose females was measured and recorded weekly during the study.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND COAGULATION: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes, carbon dioxide/oxygen
- Animals fasted: Yes, overnight prior to sample collection, but had free access to drinking water
- How many animals: all males and repeat dose females
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:at termination from all males and repeat dose females
- Animals fasted: Yes, overnight prior to sample collection, but had free access to drinking water
- How many animals: from all males and repeat dose females
- Parameters checked in table [No.1] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to initiation of test or control article administration, at the start of treatment, and weekly during the study.
- Dose groups that were examined: each repeat dose male and female
- Battery of functions tested in table [No.1] were included.

BEHAVIORAL OBSERVATION-REPEAT DOSE COMPOMENT:

- Time schedule for examinations: prior to the first exposure and during the last week of study.
- Dose groups that were examined: All males and repeat dose females
- Battery of functions tested in table [No.1] were included.

OTHER:
Fo BREEDING PROCEDURES- REPRODUCTIVE COMPONENT (see in section 7.8 Toxicity to reproduction)
Fo PARTURITION AND Fi LITTER OBSERVATIONS - REPRODUCTIVE COMPONENT (see in section 7.8 Toxicity to reproduction)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (table 2), all adult animals dying spontaneously or euthanized at scheduled necropsies were externally examined and any abnormalities were identified and correlated with antemortem findings. The carcasses were discarded without further evaluation.
Organs and tissues from all adult animals were removed, trimmed of any adherent tissues, weighed (where required) and placed in neutral buffered formalin, except for the testes and epididymides which were placed in Bouin's fixative, and the eyes which were placed in Davidson's fixative. The lungs were infused via the trachea with formalin. The urinary bladder was inflated with formalin at necropsy and examined internally after fixation.

HISTOPATHOLOGY: Yes, were performed for all males and repeat dose females in the control and high dose groups.
Microscopic examination of formalin-fixed hematoxylin and eosin-stained paraffin sections of the tissues were performed for all males and repeat dose females in the control and high dose groups. For processing of the testes, hematoxylin and period acid Schiff (PAS) staining and paraffin embedding was performed.

NEUROPATHOLOGICAL EVALUATIONS
Nervous system tissue (cervical, thoracic, and lumbar spinal cord) from female animals from the mid and high repeat dose groups (454 and 1030 mg/kg bw/day, respectively) as well as the control were sent to Experimental Pathology Laboratories, Inc. (EPL*), in Henidon, Virgina, for further processing and/or special neuropathology staining which allowed for better examination of possible nervous system effects. The sciatic nerve, when available, was processed to glycomethacrylate blocks and then sectioned and stained with Toluidine Blue or Bielschowsky for histopathologic evaluation.

Other examinations:

ORGAN WEIGHTS
Body and protocol-specified organ weights were recorded at necropsy and appropriate organ weight ratios were calculated (absolute and relative to body and brain weight) for all adult animals. Paired organs were weighed together. Organs were not weighed for animals dying spontaneously.

Statistics:

Group Pair-wise Comparisons
For each specified endpoint and for all collection intervals, Levene's test 5 was used to assess homogeneity of group variances. If Levene's test was not significant (p>0.01), Dunnett's test6 was used to compare each treatment group with the control group. If Levene's test was significant (p<0.01), comparisons with the control group were made using Welch's t-test7 with a Bonferroni correction. Results of all pair-wise comparisons were reported at the 0.05 and 0.01 significance levels. All endpoints were analyzed using two-tailed tests. Besides these tests, Log Transformation, Fisher's Exact Test, Covariate Analysis and Arcsin-Square-Root Transformation were used

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Mortality:

no mortality observed

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

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
All treated animals in the repeat dose component survived to scheduled euthanasia.
No effect of treatment with ADC was evident from the general and detailed clinical examinations of the repeat dose animals. The few findings seen among the treated animals occurred with low incidence or with similar frequency as controls. These findings (i.e. red material around the eyes or nose, sparse amounts of hair on the fore- and hindlimbs/paws) are seen commonly in this laboratory with this strain and age of animal and the occurrences in this study were considered unrelated to treatment.

BODY WEIGHT AND WEIGHT GAIN
No effect of treatment with ADC was evident from body weight data for males and females in the repeat dose component. Mean body weights for the treated animals were comparable to controls throughout the seven-week treatment study.
No effect of treatment with ADC was evident from body weight gain for males and females in the repeat dose component. Mean week-to-week body weight gain for the treated animals was comparable to controls. In the females there was a mean weight loss during the last week of study. This was seen in all groups (control and treated) and was attributed to half of the females in each group being fasted for blood collection (clinical pathology) overnight prior to euthanasia and necropsy. The remaining females in each group were necropsied the following day and were not fasted overnight.
FOOD CONSUMPTION
No effect of treatment with ADC was evident from food consumption for the males and females in the repeat dose component. Mean food consumption for the males during the two weeks prior to pairing and two weeks following completion of the pairing (mating) period was comparable to controls. Likewise, food consumption for the treated females in the repeat dose component over the entire six-week treatment period was comparable to controls.

HAEMATOLOGY
There were no adverse test article-related effects on hematology parameters evaluated. The red cell indices (MCHC, MCV, and MCH) were statistically decreased in males at 454 mg/kg/day. The differences in MCHC, MCV, and MCH were minimal, however, and not considered to be treatment related.

CLINICAL CHEMISTRY
No adverse effects were seen in clinical chemistry analytes. Few analytes in the treated groups exhibited statistically significant differences from controls, but none were considered physiologically or toxicologically relevant.

NEUROBEHAVIOUR
No effect of treatment with ADC was evident from the neurobehavioral evaluations of the repeat dose animals.

BEHAIVIOUR
Auditory Response, Corneal Reflex, Pupil Response, and Grip Strength

No effect of treatment with ADC was evident from auditory response, corneal reflex or pupil response. With the exception of one control female which did not pass the corneal reflex evaluation, all other animals (control and treated groups) passed these behavioral tests at terminal evaluation. Similarly, no effect of treatment was evident in either males or females from forelimb grip strength evaluations. Forelimb grip strength in the 150 mg/kg/day males was statistically lower than controls; however, in the absence of a similar response in males at the higher dose levels, this was considered spurious and unrelated to treatment. In both sexes, forelimb grip strength at termination was greater than at the pretest evaluation.

No effect of treatment with ADC to males was evident from hindlimb grip strength. These values in the treated males at terminal examination were comparable to controls. Grip strength in the control and treated males at terminal examination was notably lower than at pretest and differences ranged from about 43% lower in controls to 23% lower in the 1030 mg/kg/day group. During this pretest interval, hindlimb grip strength in the 150 and 1030 mg/kg/day males was statistically lower than controls and comparable to controls in the 454 mg/kg/day group.

Mean hindlimb grip strength at terminal examination among females treated at 454 and 1030 mg/kg/day was statistically significantly lower than the control group; both the mid-and high-dose groups displayed a very similar decrease. This finding appears to be spurious and not toxicologically significant because: 1) the effect was not dose responsive (grip strength was similar for the two dose groups despite the two-fold increase in dose between the mid- and high-dose groups), 2) mean hindlimb grip strength was significantly lower at termination than prior to the start of the study for all groups (treatment and control), 3) the effect was noted only in females and not in males, 4) there were no microscopic changes noted in H&E stained sections of the spinal cord or sciatic nerve or hindlimb muscle, and 5) further processing and/or special neuropathology staining of the spinal cord and sciatic nerve also did not reveal any microscopic changes that were believed to be treatment related.

Motor activity
Motor activity evaluated as horizontal activity, vertical activity, total distance, and stereotypy over a 20-minute trial was comparable to controls in the treated females and in the 150 mg/kg/day males at the terminal evaluation. Motor activity was higher than control in the 454 and 1030 mg/kg/day males during the 5-10 minute testing interval, and in the 454 mg/kg/day males over the entire 0-20 minutes of testing and in most instances these differences were statistically significant. For all other intervals (0-5, 10-15, and 15-20 minutes) motor activity for these treated males did not differ statistically from controls and was considered comparable between the groups. The increased activity in these treated males during this one five minute interval over the entire 20-minute period was not considered toxicologically meaningful since there was no clear dose response in any of the activity parameters. Mean values for each activity (horizontal, vertical, total distance, and stereotypy) were similar between the 454 and 1030 mg/kg/day groups. Likewise, cumulative evaluations over the entire 20-minute testing period were also not dose responsive, and only differed statistically from control at the 454 mg/kg/day dose level. During the pretest evaluation, motor activity for the treated males and females (all groups) was comparable to controls.
Emotionality
No effect of treatment with ADC was evident from the emotionality evaluations conducted during the first five minutes of the motor activity testing. The parameters recorded during these evaluations (intervals of urination and counts of defecation, grooming, backing and rearing) for the treated groups were comparable to controls.

ORGAN WEIGHTS
No test article-related organ weight changes were noted in rats of either sex that received ADC in the repeat dose phase of the study.
Absolute mean prostate weights and relative prostate weights to body and brain weights were statistically significantly increased in male rats that received 454 mg/kg/day and 1030 mg/kg/day ADC compared to controls. However, there were no corresponding microscopic changes in prostates of males in these groups. The prostate weight increase was not considered to be toxicologically significant. Absolute mean heart weight and relative heart weight to body and brain weights were statistically significantly increased in female rats that received 1030 mg/kg/day ADC compared to controls, but males were not affected and there were no corresponding microscopic changes in hearts. The heart weight increase was not considered to be toxicologically significant.

GROSS PATHOLOGY
No test article-related macroscopic changes were observed in rats of either sex.

HISTOPATHOLOGY: NON-NEOPLASTIC
No test article-related microscopic changes were noted in rats of either sex that received repeated doses of ADC. Microscopic changes observed were typical of those commonly found in animals of the same strain and age and were considered to be incidental.

OTHER FINDINGS
NEOROPATHOLOGICAL EVALUATIONS
The purpose of these additional neuropathology evaluations was to determine if the decrease in hindlimb grip strength in mid-and high dose females was associated with any neuropathological findings. Cervical, thoracic and lumbar spinal cord tissues were submitted for histopathological evaluation
In the neuropathological evaluations of selected nervous tissues conducted by EPLS (Report No: 139-019), minimal axonal degeneration characterized by either the presence of digestion chambers and /or swollen axons (spheroids) was observed in one level of spinal cord from one high-dose (1030 mg/kg/day) female and from one mid-dose (454 mg/kg/day) female. The sciatic nerve of one high-dose female and two mid-dose females also had minimal axonal degeneration. Similar minimal changes were not observed in the control animals. These minimal lesions were considered well within the range of what could occur as an incidental background finding. Thus, it was concluded that since there was no dose response, the changes seen were probably incidental background findings unrelated to test article treatment.

Dose descriptor:

NOAEL

Effect level:

>= 1 030 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: There were no toxicologically significant treatment related effects in all parameters tested

Critical effects observed:

not specified

Conclusions:

In this rat dermal repeat dose toxicity study with a reproduction/developmental toxicity component, the No-Observable-Adverse-Effect Level (NOAEL) of the test article diallyl diglycol carbonate, for parental toxicity was 1030 mg/kg/day, the highest dose level evaluated.

Executive summary:

This study was conducted in accordance with OECD Guideline 422, which is comprised of two components, a repeat dose toxicity study with neurobehavioral evaluations and a reproduction/developmental toxicity screening study (see section 7.8 Toxicity to reproduction for summary).

The purpose of the repeat dose toxicity component was to provide information on possible target organ effects and the potential for neurobehavorial effects arising from repeated exposures. Each repeat dose group contained ten male and ten female Sprague-Dawley rats [Crl: CD® (SD)IGS BR]. The ten male animals of each repeat dose group were also utilized for the reproductive/developmental component of the study. Animals were administered dermally once daily via occlusion for 6 hours at dose levels 150, 454, and 1030 mg/kg/day for at least 42 consecutive days. The control animals received 0.9% Sodium Chloride, USP, at a volume of 0.9 mL/kg for the same duration as the treated animals. Observations of the animals included clinical signs, neurobehavioral observations (repeat dose component only), dermal evaluation, body weights, and food consumption. Evaluations for motor activity, emotionality, and other behavioral observations, including auditory response, grip strength, pupil reflex, and corneal reflex, were conducted for all repeat dose animals, pretest and prior to scheduled terminal euthanasia (after seven weeks of treatment). Blood collections for clinical pathology evaluations were conducted at study termination. Complete necropsies were performed on all animals (repeat dose and reproductive components) and organs and tissues were collected, weighed, and preserved. Organs and tissues from control and high-dose animals in the repeat dose component were examined microscopically. Nervous system tissues (cervical, thoracic, and lumbar spinal cord and sciatic nerve) from female animals from the mid and high dose groups (454 and 1030 mg/kg bw/day) as well as the control were evaluated for neuropathology.

No effect of treatment was evident from mortality, clinical evaluations, neurobehavioral evaluations, dermal evaluations, body weights, food consumption, hematological evaluations, serum clinical chemistry, organ weights, macroscopic, or microscopic evaluations. Decreased hindlimb grip strength as well as axonal dgenerations in spinal cord and sciatic nerve were noted at termination for female animals in the 454 and 1030 mg/kg/day dose groups (Table 2. in "Remarks on results"). These findings appear to be spurious and not toxicologically significant.

Thus, in this rat dermal repeat dose toxicity study with a reproduction/developmental toxicity component, the No-Observable-Adverse-Effect Level (NOAEL) of the test article diallyl diglycol carbonate, for parental toxicity was 1030 mg/kg/day, the highest dose level evaluated.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

Oral

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.

 

Dermal

The study "Diallyl Diglycol Carbonate: combined repeated dose rat dermal toxicity study with a reproduction/developmental toxicity screening test (OECD 422)" is taken for risk characterization of repeated dermal exposures of the test material. This study was comprised of two components, a repeated dose toxicity study with neurobehavioral evaluations and a reproduction/developmental toxicity screening study. The purpose of the repeated dose toxicity component was to provide information on possible target organ effects and the potential for neurobehavorial effects arising from repeated exposures. Each repeated dose group contained ten male and ten female Sprague-Dawley rats [Crl: CD® (SD)IGS BR]. The ten male animals of each repeated dose group were also utilized for the reproductive/developmental component of the study. Animals were administered dermally once daily via occlusion for 6 hours at dose levels 150, 454, and 1030 mg/kg/day for at least 42 consecutive days. The control animals received 0.9% Sodium Chloride, USP, at a volume of 0.9 mL/kg for the same duration as the treated animals. Observations of the animals included clinical signs, neurobehavioral observations (repeated dose component only), dermal evaluation, body weights, and food consumption. Evaluations for motor activity, emotionality, and other behavioral observations, including auditory response, grip strength, pupil reflex, and corneal reflex, were conducted for all repeat dose animals, pretest and prior to scheduled terminal euthanasia (after seven weeks of treatment). Blood collections for clinical pathology evaluations were conducted at study termination. Complete necropsies were performed on all animals (repeated dose and reproductive components) and organs and tissues were collected, weighed, and preserved. Organs and tissues from control and high-dose animals in the repeated dose component were examined microscopically. Nervous system tissues (cervical, thoracic, and lumbar spinal cord and sciatic nerve) from female animals from the mid and high dose groups (454 and 1030 mg/kg bw/day) as well as the controls were evaluated for neuropathology.

No effect of treatment was evident from mortality, clinical evaluations, neurobehavioral evaluations, dermal evaluations, body weights, food consumption, hematological evaluations, serum clinical chemistry, organ weights, macroscopic, or microscopic evaluations. Decreased hindlimb grip strength as well as axonal degenerations in spinal cord and sciatic nerve were noted at termination for female animals in the 454 and 1030 mg/kg/day dose groups. These findings appear to be spurious and incidental background findings and not toxicologically significant.

Thus, in this rat dermal repeat dose toxicity study with a reproduction/developmental toxicity component, the No-Observable-Adverse-Effect Level (NOAEL) of the test article diallyl diglycol carbonate, for parental toxicity was 1030 mg/kg/day, the highest dose level evaluated.

In a previous supporting range-finding 14-day dermal study in rats (MPI, 2005), animals were administered dermally with diallyl diglycol carbonate at 150, 300 and 600 mg/kg/day. No mortality was observed during the study. No effect of treatment at all dose levels was evident from clinical examinations, dermal irritation evaluations, food consumption, macroscopic evaluations, or organ weights. A slight reduction in body weight and body weight gain for males in the highest dose group was observed. Therefore, No-Observed-Effect-Level (NOEL) for males was 300 mg/kg/day and for females was 600 mg/kg/day, the highest dose level evaluated.

In an older "Fourteen-Day Range Finding Study in Rats with Diallyl Diglycol Carbonate" (Will Research Laboratories, 1980a, dated 1980 -01 -21), no signs of toxicity were observed in any of tested animals in the lower dose groups (91.4 and 457.2 mg/kg bw). There were problems with the dosing of the high dose 2286 mg/kg bw. There was unabsorbed test material on the application area. In the next "Fourteen-Day Range Finding Study in Rats with Diallyl Diglycol Carbonate" (Will Research Laboratories, 1980b, dated 1980 -08 -0) only one dose level of 2000 µL/kg bw was tested in order to confirm findings observed at this dose level in the previous range finding study. In this study, all animals had a collar placed on the neck to prevent oral ingestion for the entire study. The excess compound was removed from the hair coat by the daily wiping of the hair and excess compound was also wiped from the cage surfaces. The findings were the same as in a previous study. The main toxic effect observed was a significant decrease in body weight in male rats only. This was supported by some decreases in food consumption.

The outcomes of the supporting studies confirm the accuracy of the established NOAEL in the Combined Repeated Dose rat Dermal Toxicity Study with a Reproduction/Developmental Screening Test.

Justification for classification or non-classification

After repeated exposure of rats to the registered substance over a period of 90 days in a highly reliable GLP OECD 408 guideline study signs of systemic toxicity were revealed. Based on the liver finding, as described in detail above, the criteria for classification in Category 2 for target organ toxicity after repeated exposure are met. Adverse effects in the liver were also reported in F0 and F1 animals of a highly reliable EOGRTS (OECD 443, GLP), which further confirms the specific target organ toxicity evoked by the registered substance.

Therefore, classification of the registered substance for Specific Target Organ Toxicity - Repeated Exposure Category 2 according to Regulation (EC) No 1272/2008 is warranted.