Oxybis(methyl-2,1-ethanediyl) diacrylate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Mar - Aug 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliance

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed on August 2019

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)] diacrylate

EC Number:

256-032-2

EC Name:

(1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)] diacrylate

Cas Number:

42978-66-5

Molecular formula:

C15 H24 O6

IUPAC Name:

(1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)] diacrylate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: ReachCentrum SA
- Batch number of test material: 180003P040
- Expiration date of the lot/batch: 31. Dec 2018
- Purity/Composition: 100% (UVCB)
- Appearance: clear, colorless liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Stability under storage conditions: until 31. Dec 2018
- Stability of the test substance in the vehicle: Stability for at least 24 hours at room temperature protected from light, stability for at least 8 days in the refrigerator, and stability of 0.5 mL samples for at least 3 weeks in the freezer (≤ -15°C) is confirmed over the concentration range 1 to 200 mg/mL (solutions)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 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 daily as a solution and dosed within 6 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han), outbred, SPF-Quality

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. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source.
This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks (males), 13 weeks (females)
- Weight at study initiation: males: 251- 322 g; females: 198- 263 g
- Housing: individually (females during the post-mating phase and lactation phase with the pups) and grouping (pretest, males during the post-amting phase
- Diet: ad libitum; pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum
- Acclimation period: for 5 days prior to start of the pretest period (females) or 5 days before the commencement of dosing (males).

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water is performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40-70 (daily mean relative humidity of 36 to 60%)
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
Test item dosing formulations (w/w) were homogenised to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item.

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.
- Concentration in vehicle: 8, 25, 75 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:

Analyses were performed using a validated analytical procedure. Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.

Duration of treatment / exposure:

males: 29 days; females: 50-62 days
The duration of treatment covered a 2-week premating period and mating in both sexes as well as entire gestation and the duration of pregnancy and at least 14 days after delivery,
up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 40-53 days.

Frequency of treatment:

daily (7d/week)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

40 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

125 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

375 mg/kg bw/day (nominal)

No. of animals per sex per dose:

- in total 10 animals/ sex/ dose
- 5 animals /sex/ dose were selected for functional tests (males only), clinical pathology, collection of full list of organs/tissues at macroscopic examination, organ weights (full list) and histopathology (full list)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 16-day dose range finder with oral gavage administration of the test item.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
F0 animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Pups were observed daily for general health/mortality. The number of live and dead pups were determined on PND 1 and daily thereafter.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily (F0 and pubs)

BODY WEIGHT: Yes
- Time schedule for examinations: F0: First day of treatment (prior to dosing) and weekly thereafter (after dosing); F1: Live pups were weighed individually on PND 1, 4, 7 and 13.
- Body Weight Gains: Calculated against the body weight on Day 1 (premating, mating and lactation periods) or Day 0 (postcoitum period).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Time schedule for examinations: quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
- Relative Food Consumption Calculated against the body weight for scheduled intervals.

WATER CONSUMPTION: not quantitative
- Subjective appraisal was maintained during the study

HAEMATOLOGY: Yes, F0 animals
- Time schedule for collection of blood: day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, males only (maximum of 24 hours)
- How many animals: 5/sex/group
- Parameters checked in table 1 were examined.

COAGULATION
Blood plasma of F0 animals was analyzed for Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, males only (maximum of 24 hours)
- How many animals: 5/sex/group
- Parameters checked in table 2 were examined.

OTHER:
Functional observational battery (FOB): Males only
- Functional tests were performed on the selected 5 males (F0) during Week 4 of treatment (after dosing, after completion of clinical observations)
- Examined parameters:
• Hearing ability
• Pubillary reflex
• Static righting reflex
• Fore- and hind-limb grip strength
• Locomotor activity

Estrous cycle:
- Daily vaginal lavage was performed for all females (F0) beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.

Female reproduction and delivery data
From the mating period onwards, the following parameters were recorded for each female (F0): male number paired with, mating date, confirmation of pregnancy and delivery day.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care

Thyroid hormones
- Time schedule for collection of blood: All F0 animals on scheduled necropsy, PND 14-16 and PND 4 for 2 pubs per litter
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: males only ( mximum of 24 h)
- How many animals: All F0 animals, 2 pubs per litter on PND 4 and PND 4-16
- For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no adverse changes in T4 were noted in F0- males, no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight were recorded
- Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16

OTHER:
Sex was externally determined for all pups on PND 1 and 4.
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
All male pups in each litter were examined for the number of areola/nipples on PND 13.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 3 and 4, Organ weights)

HISTOPATHOLOGY: Yes (see table 5 for collected tissue)
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
- Selected animals: Tissues identified in Text Table 5 (except animal identification, aorta, nasopharynx, esophagus, harderian
gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue).
- Males that failed to sire (except for males which were selected), females that failed to deliver pups and females with total litter loss: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.
- Females with total litter loss: Mammary gland.
- Remaining animals: Gross lesions/masses.

Statistics:

Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation seen after dosing among animals of all dose groups was not considered toxicologically relevant, 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 related to taste of the test item rather than a sign of systemic toxicity.
Further observed clinical signs affecting the skin/fur (alopecia, scabs), the eye (Chromodacryorrhoea) and breathing (rales) were observed.

Mortality:

no mortality observed

Description (incidence):

One female of the control group (no. 49) was euthanized on Lactation Day 1, as she had a total litter loss (at first litter check she had only dead pups).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight gain was increased in males in the second week of treatment after dosing of 125 and 375 mg/kg, and in the fifth week of treatment after dosing with 375 mg/kg. These changes in body weight gain were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment and values were within the historical control range

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significant change in relative food consumption in 40 mg/kg females during post-coitum Days 17-20 was considered to be unrelated to treatment, since no trend was apparent regarding dose and duration of treatment.

Food efficiency:

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A decreased number of reticulocytes was observed in females at 125 mg/kg, which was considered unrelated to administration of the test item due to absence of a dose-related trend response.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males: The following statistically significant increases were noted for treated males (relative changes in mean values as compared to the concurrent control group are indicated between parentheses):
-increased total protein at 375 mg/kg (6%)
-increased albumin at 375 mg/kg (6%)
-increased calcium at 375 mg/kg (4%)
-increased urea at 40 and 375 mg/kg (27% and 30%, respectively)
The changes in total protein, albumin and calcium were minimal and remained within the historical control range. No dose related trend was observed for the increase in urea and values remained within the historical control range. In addition, a slight increase in sodium was observed at 125 mg/kg (1%), which occurred in the absence of a dose related trend.

No treatment-related changes were noted in clinical chemistry parameters in females

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 higher liver weights (absolute and relative to body weights) were noted in the 375 mg/kg/day group males.
There were no other test item-related organ weight changes.
The significant relative prostate gland weight decrease and liver weight increase in the 40 mg/kg/day treated males was considered incidental and not related to treatment in absence of a dose-related trend.

for details see table 6

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related irregular surface was observed in the (fore)stomach in 2/10 males treated at 125 mg/kg/day and in 10/10 males and 2/10 females treated at 375 mg/kg/day.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Watery fluid in the uterus, found in one control female and 3 females treated with 125 mg/kg, is related to a stage in the estrous cycle and is a normal finding.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the (fore)stomach of males and females and the liver and kidneys of males.

Stomach, squamous cell hyperplasia was present in 2/5 males starting at 125 mg/kg/day up to marked degree and in females at 375 mg/kg/day up to moderate degree. This correlated with the macroscopic irregular surface.
Stomach, ulcer forestomach was present in males starting at 125 mg/kg/day at minimal degree.
Stomach, inflammation forestomach was present in males starting at 125 mg/kg/day up to moderate degree.

Liver, hepatocellular hypertrophy was present in males treated at 375 mg/kg/day at minimal degree. This correlated with the increased liver weight. Due to the absence of any indicators of cellular degeneration. The changes in the liver were not considered adverse at current severities.

Kidneys, an increased incidence and severity of hyaline droplet accumulation was present in males treated at 375 mg/kg/day up to slight degree. The increased hyaline droplet accumulation in the male kidneys was not accompanied by indicators of tubular damage and therefore this was considered to be nonadverse.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

- Functional observation parameters were not considered to be affected by treatment in males up to 375 mg/kg.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Forelimb and hind limb grip strength was similar between control and treated animals. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
- Coagulation parameters (prothrombin time and activated partial thromboplastin time) of treated rats were considered not to have been affected by treatment.
-Serum levels of T4 in F0 males were increased at 125 and 375 mg/kg (35% and 37% increase in mean values compared to concurrent control, respectively). These values remained within the historical control range and the control value was on the lower limit of this range. In addition, no effect was observed in respect to thyroid weight, therefore, this effect was considered not toxicologically relevant.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

375 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects up to and including the highest tested dose

2

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

The various analyses confirmed

- 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 formulation.

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

Table 6: Mean Percent Liver Weight Differences from Control Groups

Dose level in mg/kg bw/d	Males			Females
	40	125	375	40	125	375
LIVER
absolute	13	4	31**	-7	-4	1
Relative to bodyweight	9*	5	25**	-6	-1	0

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

Table 7: Summary Test Item-Realted Microcopic Findings-(fore)stomach

Dose level in mg/kg bw/d	Males				Females
	0	40	125	375	0	40	125	375
STOMACH^a	5	5	5	10	5	5	5	5
Hyperplasia squamous cell
Slight	-	-	-	-	-	-	-	1
Moderate	-	-	2	6	-	-	-	1
Marked	-	-	-	4	-	-	-	-
Ulcer forestomach
minimal	-	-	1	2	-	-	-	-
Inflammation forestomach
Minimal	-	-	1	6	-	-	-	-
Moderate	-	-	1	-	-	-	-	-

a = Number of tissues examined from each group

Table 8 Summary Test Item-Related Microscopic Findings – Liver and Kidneys – Males

	Males
Dose level in mg/kg bw/d	0	40	125	375
LIVER^a	5	5	5	5
Hepatocellular hypertrophy
minimal	-	-	-	4
KIDNEY^a	5	5	5	5
Hyaline droplet accumulation
minimal	1	1	2	3
slight	-	-	-	2

a= Number of tissues examined from each group

Applicant's summary and conclusion

Conclusions:

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) were established:
Parental local NOAEL: 40 mg/kg (based on findings in the (fore)stomach)
Parental systemic NOAEL: at least 375 mg/kg due to the absence of adverse toxicity in the study for both sexes.

Executive summary:

Wistar Han rats were treated with the test item by daily oral gavage at dose levels of 40, 125 and 375 mg/kg according to OECD 422 and in compliance with GLP. The rats of the control group received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-62 days). Females that failed to deliver pups were treated for 40-53 days.

Parental toxicity was observed in the (fore)stomach of males from 125 mg/kg and in females at 375 mg/kg.

These changes consisted of ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females.

Other treatment-related but non-adverse changes were observed in the liver at microscopic examination. An absolute increase of 31% and a relative increase of 25% in liver weight was observed at dose 375 mg/kg. At microscopic examination, hepatocellular hypertrophy in the liver was observed at minimal severity and was in the absence of any indicators of cellular degeneration. The changes in the liver were not considered adverse at current severities. In the kidneys an increase in hyaline droplet accumulation was recorded in males which was considered to likely represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules (Alden et al., 1991). This male rat specific protein is not present in female rats nor in higher mammals, including man (Sahota et al., 2013). The increased hyaline droplet accumulation in the male kidneys at 375 mg/kg/day was not accompanied by indicators of tubular damage and therefore this was considered to be nonadverse.

Functional observations were not performed for females and therefore, possible treatment related effects on the functional parameters could not be evaluated.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations (males), body weight, food consumption, clinical laboratory investigations (including male T4 thyroid hormone levels), macroscopic examination and organ weights).

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, no systemic no-observed-adverse-effect level (NOAEL) were established under the conditions of this study. The Parental local NOAEL of 40 mg/kg is based on the findings in the (fore)stomach. Due to the absence of systemic toxicity up to and including the highest tested dose of 375 mg/kg bw/d no systemic NOAEL could be derived.