reaction products of 1,4-butanediol with 1,3-chloro-2,3-epoxypropane, esters with prop-2-enoic acid

Endpoint summary

Description of key information

A 90-day repeated toxicity study (CRL 2021, OECD 408) is available on butane-1,4-diylbis(oxy-2-hydroxypropane-3,1-diyl) bisacrylate.

Based on this study, the no observed adverse effect level (NOAEL) for general systemic toxicity was 30 mg/kg bw/d in rats.

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:

9 June 2020 - 23 September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

25 June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl CD® (SD) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Italia, Calco, Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Weight at study initiation: 192.3 - 253.8 g (males) and 138.2 - 181.7 g (females)
- Housing: The animals were group-housed in two from the same sex and group in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm²) containing autoclaved sawdust (Le comptoir des sciures, Meyzieu, France). Animals were isolated where experimental procedure dictated. This was documented in the study file. Each cage contained rat hut and nylabone for environmental enrichment.
- Diet: ad libitum, SSNIFF rat/mouse pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum, bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: 14 days

CONTAMINANT ANALYSES
No contaminants were present in the diet, drinking water or sawdust at levels which could have been expected to interfere with, or prejudice, the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): approx. 8-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 9 June 2020 To: 23 September 2020

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected since it is a route of administration which is requested by the Regulatory Authorities for this type of test item.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were maintained under delivery conditions (at room temperature and protected from light) throughout the administration procedure.
The control dose formulations were stirred just before administration, the test item dose formulations for at least 30 minutes before administration, and then throughout the whole administration procedure.
The test item dose formulations were administered within 4 hours after the end of their preparation.

VEHICLE
- Concentration in vehicle: 2, 6, 20 mg/mL
- Amount of vehicle: 5 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On three occasions during the study (in Weeks 1, 6 and 12) a sample was taken from control and test item dose formulations and analyzed using the validated method. The samples were analyzed with an LC/MS-MS.
Acceptance criterion: Measured concentration = nominal concentration ± 15%

Duration of treatment / exposure:

at least 13 weeks

Frequency of treatment:

daily

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected in agreement with the Sponsor, based on the results of a previous Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test (OECD 422) in Wistar Rats (Gavage), in which the test item was administered by daily oral gavage to male and female rats at dose levels of 50, 150 or 500 mg/kg bw/day. Because of clinical findings and the premature death of 3 male animals at the high dose level, the dose was reduced at 300 mg/kg bw/day from Day 6 of the study.
At 500/300 mg/kg bw/day, parental toxicity consisted of premature deaths for 6 males and 3 females (one female was found dead and one female was prematurely euthanized for delivery difficulties at the end of the gestation period, and one female died during the lactation period), clinical signs (i.e. hypersalivation, breathing difficulties and/or piloerection in males and females), reduced body weight and body weight gain for males and affected hematological parameters (i.e. increased neutrophil count and decreased lymphocyte count) in males. Histopathological changes were seen in the forestomach (i.e. erosion/ulcer and/or related squamous hyperplasia or inflammation/edema in 9/10 males and 10/10 females) and in the stomach (i.e. erosions or ulcers in 3/10 males).
At 150 mg/kg bw/day, there were breathing difficulties in males and females, hypersalivation and/or transient piloerection in isolated males and females and reduced body weight in males on mating Day 8. Histopathological changes were seen in the forestomach (i.e. erosion/ulcer and/or related squamous hyperplasia or inflammation/edema in 9/10 males and 7/10 females) and in the stomach (i.e. erosions or ulcers in 1/10 females).
At 50 mg/kg bw/day, hypersalivation was noted in males and females. Histopathological changes were seen in the forestomach (i.e. erosion/ulcer and/or related squamous hyperplasia or inflammation/edema in 6/10 males and 1/10 females).
Pathological findings observed at all dose levels were related to the irritating potential of the test item.
Based on the above-mentioned results and the histopathological changes observed at 150 mg/kg/day in most animals after 4-8 weeks of exposure, 100 mg/kg bw/day was selected as the high-dose level in the 13-week exposure study. The low- and intermediate-dose have been selected using a ratio representing approximately a 3-fold interval (i.e. 30 and 10 mg/kg bw/day).
- Fasting period before blood sampling for clinical biochemistry: at least 14 hours but not exceeding 18 hours

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Each animal was checked for mortality and morbidity at least once a day during the acclimation period and at least twice a day during the treatment period, including weekends and public holidays.
- Each animal was observed for the recording of clinical signs: once a day before the beginning of the treatment period, at least once a day during the treatment period, after completion of treatment of the last animal in the animal room.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once before the beginning of the treatment period, at least once a week during the treatment period.
- Scabs and alopecia localized on the cervical region (neck) were observed in one female of group 3 in Week 6. These lesions required medical treatment which consisted of the application of an antiseptic/cicatrizing agent for 5 days (containing essential oils, Cothivet®, Vetoquinol, France).

BODY WEIGHT: Yes
- Time schedule for examinations: at least once a week before the beginning of the treatment period, on the first day of treatment, and at least once a week until the end of the study.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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: Yes
- Time schedule for examinations: on all animals, before the beginning of the treatment period, and on control and high-dose animals on one occasion during Week 13.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13 before daily treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked in table were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13 before daily treatment
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked in table were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: week 13
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: T3, T4 and TSH

URINALYSIS: Yes
- Time schedule for collection of urine: week 13 during overnight period of at least 14 hours
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table were examined.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

MONITORING OF ESTROUS CYCLE:
The estrous cycle stage was determined daily from a fresh vaginal lavage (stained with methylene blue), on the day of euthanasia and daily during the 4 days before euthanasia.
Only individual estrous cycle data were presented in the study report. No statistical analyses were performed on those data.

Sacrifice and pathology:

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

ORGAN WEIGHTS:
The body weight of each animal was recorded before euthanasia at the end of the treatment period. The organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.

HISTOPATHOLOGY:
- For all study animals, the tissues specified in the Tissue Procedures Table were preserved in 10% buffered formalin (except for the eyes and optic nerves and Harderian glands, and the testes and epididymides which were fixed in Modified Davidson's Fixative).
- A microscopic examination was performed on: all tissues listed in the Tissue Procedure Table for the control and high-dose animals (groups 1 and 4), all macroscopic lesions, forestomach, and spleen (females) for all low- and intermediate dose animals (groups 2 and 3).
- In addition, testicular staging was performed for control and high-dose males (groups 1 and 4). A detailed examination of the testes was performed, using a thorough understanding of tubule development through the different stages of the spermatogenic cycle. Transverse sections of the testes were stained with hematoxylin-PAS in order to detect retained spermatids, missing germ cell layers, multinucleated giant cells or sloughing of spermatogenic cells into the lumen, etc.

BONE SMEARS:
Two bone marrow smears were prepared from the femoral bone (at necropsy) of all animals euthanized on completion of the treatment period. In absence of abnormalities at the haematological investigations or histopathological examination requiring a bone marrow differential cell count determination, the smears were archived without further investigations.

Statistics:

Provantis software was used to perform the statistical analysis of body weight, food consumption, haematology, coagulation, blood biochemistry, thyroid hormones and urinalysis. See illustration for sequence followed.
PATHDATA software was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01) according to the following sequence:
- Non parametric testing: Test for group differences Kruskal-Wallis if > 2 groups; Dunn test if > 2 groups Wilcoxon test if 2 groups
- Parametric testing: Test for group differences in means One-way analysis of variance if > 2 groups; Dunnett test if > 2 groups t-test if 2 groups

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The only test item-related clinical sign observed during the study was minimal to severe hypersalivation in 2/10 males and 2/10 females at 30 mg/kg bw/day and in 9/9 males and 3/10 females at 100 mg/kg bw/day. While observed at a low frequency (up to 6 occasions) in males and females at 30 mg/kg bw/day and in females at 100 mg/kg bw/day, hypersalivation was reported almost throughout the treatment period in most males at 100 mg/kg bw/day. Hypersalivation, a common observation when a test item is administered by gavage, had no impact on the general condition of the animals and was therefore considered not adverse. This was considered likely to be related to the irritating potential of the test item.
All other clinical signs observed during the study (i.e. reflux at dosing, chromodacryorrhea, chromorhynorrhea, soiled coat, alopecia, scabs, nodule, thin or erected fur, bent tail, half-closed eyes and/or short, broken or abnormal growth of teeth) were considered incidental as they were occasional, observed with no dose-relationship and/or can commonly be encountered in laboratory rats dosed by gavage.

Mortality:

mortality observed, treatment-related

Description (incidence):

There was one unscheduled death during the study.
One male dosed 100 mg/kg bw/day died on Day 85 (during the ophthalmological examination). Except for repeated hypersalivation and occurrences of loud breathing on Days 73, 74 and 76, no clinical signs were observed before death. Microscopically, test item-related moderate multifocal erosion/ulceration (down to submucosa and with bacteria) along with marked inflammation/oedema (mixed inflammatory cells infiltrates in the submucosa) were noted in the forestomach, and correlating with many raised white masses up to 0.2 cm in diameter at necropsy. These changes most likely contributed to the death of this animal, which was considered as test item related. Moderate squamous cells hyperplasia and hyperkeratosis in the forestomach was considered a reaction to ulceration.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item-related effects on body weights or body weight gains during the study.
Any differences between control and test item-treated groups, including those that were statistically significant, were transient, observed sporadically with no dose-relationship and/or were consistent with normal biological variations, and were therefore not considered to be test item-related.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item-related effects on food consumption during the study.
Any differences between control and test item-treated groups, including those that were statistically significant, were transient, observed sporadically with no dose-relationship and/or were consistent with normal biological variations, and were therefore not considered to be test item-related.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related ophthalmological findings at the end of the treatment period in animals at 100 mg/kg bw/day. Animals at 10 and 30 mg/kg bw/day were therefore not examined at the end of the treatment period.
Hyaloid remnants were observed in the right eye vitreous body of one male at 100 mg/kg bw/day but as they were already observed pre-test, as well as in one control male, these findings were considered to be incidental and not related to the test item.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test item-related effects on haematology parameters at the end of the treatment period.
Any differences between control and test item-treated groups were not statistically significant and not dose-related, of minimal amplitude and/or consistent with normal biological variations, and were therefore not considered to be test item-related.
There were no changes in prothrombin time at the end of the treatment period.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on blood biochemistry parameters at the end of the treatment period.
Any differences between control and test item-treated groups, including those that were statistically significant, were not dose-related, of minimal amplitude and/or consistent with normal biological variations, and were therefore not considered to be test item-related.
Specifically, the statistically significantly higher mean total bilirubin concentration in males at 100 mg/kg bw/day compared to controls (1.26 µmol/L vs. 0.80 µmol/L in controls) was consistent with normal biological variations. The non statistically higher total bilirubin concentration, alanine aminotransferase, aspartate aminotransferase activity and/or biliary acids concentration in females at 10 or 30 mg/kg bw/day were isolated, related to one or two females and not dose-related; they were all considered incidental.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Higher TSH (mainly in males at 10 mg/kg bw/day due to a high interindividual variability), T3 and T4 levels were observed in test item-treated males when compared to controls, reaching statistical significance in T4 levels at 100 mg/kg bw/day. These changes which were within the HCD, were observed with no dose relationship and/or had no histopathological correlates and were therefore not considered to be test item-related.
There were no relevant changes on thyroid hormones levels in females at the end of the treatment period.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test item-related findings on quantitative and qualitative urinary parameters at the end of the treatment period.
Any differences between control and test item-treated groups were not statistically significant and not dose-related, of minimal amplitude and/or consistent with normal biological variations and were therefore not considered to be test item-related.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no organ weight changes related to the test item administration at the end of the treatment period.
The statistical organ weight changes for kidneys in females were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates and were not consistent for the sexes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related macroscopic observations were noted in the forestomach at 100 mg/kg bw/day in both sexes.
At 100 mg/kg bw/day, the forestomach showed white masses and/or was gelatinous in both sexes. It correlated with microscopic inflammation/edema and/or squamous cell hyperplasia.
None of the remaining macroscopic findings were test item-related because they were consistent with spontaneously occurring findings described in the literature, the findings were distributed randomly among groups, or their appearance was similar to findings found in controls.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic observations were observed in the forestomach from 30 mg/kg bw/day at the end of the treatment period.
From 30 mg/kg bw/day in males and at 100 mg/kg bw/day in females, minimal to moderate erosion/ulcer, inflammation/oedema (mixed inflammatory cells infiltrates in the submucosa) and squamous cell hyperplasia were observed in the forestomach. Incidence and/or severity grades were higher in males when compared to females.
There were no test item-related changes noted in the testes or epididymides at microscopic evaluation. Unilateral testicular hypoplasia was noted in a single male at 100 mg/kg/day; this is a congenital lesion of the testes which can be observed in controls rats and was therefore considered as incidental and unrelated to the test item administration.
No test item-related microscopic changes were observed in the thyroid glands. The few microscopic findings observed in test item-treated animals (minimal infiltrate of mononuclear inflammatory cells and ectopic thymic tissue both observed in isolated males at 100 mg/kg bw/day) were of those commonly seen in the thyroid glands of control rats and were considered to be incidental.
In females at 100 mg/kg bw/day, minimal to slight increased lymphoid cellularity (germinal centers) was observed in the mandibular lymph nodes of 2/10 animals. In view of the low incidence and magnitude of this change, it was considered as incidental and not test item-related.
None of the remaining microscopic findings were considered as test item-related since they were consistent with spontaneously occurring findings described in the literature, the findings were distributed randomly among groups, or their appearance was similar to findings found in controls.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

MONITORING OF ESTROUS CYCLE:
There were no test item-related effects on estrous cycle during the 5 days of evaluation at the end of the treatment period, with comparable data between control and test item-treated females.

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Dose formulation analysis

The test item concentrations in the administered dose formulations analyzed in Weeks 1, 6 and 12 remained within an acceptable range of variations (-4.7% to +6.5%) when compared to the nominal values (± 15% of the nominal concentrations).

No test item was observed in the control dose formulations.

 

 

Incidence and Severity of Selected Microscopic Findings in the Forestomach at the End of the Treatment Period

Organ Finding	Male				Female
Dose level (mg/kg bw/day)	0	10	30	100	0	10	30	100
Num. of exam. animals/Num. of treated animals	10/10	10/10	10/10	9/10	10/10	10/10	10/10	10/10
Forestomach
Erosion/ulcer
Minimal (Grade 1)	-	-	2	-	-	-	-	2
Slight (Grade 2)	-	-	-	1	-	-	-	-
Inflammation/Edema
Minimal (Grade 1)	-	-	1	-	-	-	-	-
Slight (Grade 2)	-	-	1	2	-	-	-	2
Moderate (Grade 3)	-	-	-	5	-	-	-	1
Hyperplasia; squamous cell
Minimal (Grade 1)	-	-	1	3	-	-	-	3
Slight (Grade 2)	-	-	1	3	-	-	-	2
Moderate (Grade 3)	-	-	-	3	-	-	-	-

-: not observed.

Conclusions:

In a 90-day repeated dose toxicity study the oral administration of the test item in rats induced erosion/ulcer, inflammation/oedema and secondary squamous cell hyperplasia in the forestomach from 30 mg/kg bw/day in males and at 100 mg/kg bw/day in females. These changes were considered as adverse at 100 mg/kg bw/day in both sexes and contributed to the premature death of one male at 100 mg/kg bw/day. Based on these adverse findings at 100 mg/kg bw/day, the No-Observed-Adverse-Effect Level (NOAEL) in this study was considered to be 30 mg/kg bw/day.

Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item,
1,4-butanediylbis[oxy(2-hydroxy-3,1-propanediyl) diacrylate, following daily oral administration (gavage) to rats for 13 weeks.
Four groups of ten males and ten females Sprague-Dawley rats were treated daily by the oral route (gavage) with the test item, 1,4-butanediylbis[oxy(2-hydroxy-3,1-propanediyl) diacrylate, at dose levels of 0, 10, 30 or 100 mg/kg/day for 13 weeks (i.e. 91 or 92 days according to the necropsy schedule). The test item was administrated as an emulsion in the vehicle (corn oil) at a constant dosage volume of 5 mL/kg/day.
The following parameters and endpoints were evaluated in this study: survival, clinical signs, body weights, body weight gains, food consumption, ophthalmology, clinical pathology (hematology, coagulation, blood biochemistry, and urinalysis as well as thyroid hormones), estrous cycle, gross necropsy findings, organ weights, and histopathologic examinations.
The actual test item concentrations in the analyzed dose formulations were within an acceptable range of variations (-4.7% to +6.5%) when compared with the nominal values (nominal ± 15%).
The unscheduled death of one male at 100 mg/kg/day on Day 85 was attributed to the test item and considered to be related to adverse moderate erosion/ulceration and marked inflammation/edema in the forestomach.
Test item-related clinical signs in surviving animals consisted of minimal to severe
hypersalivation, observed at a low incidence and frequency in males and females at 30 mg/kg/day and females at 100 mg/kg/day, and at a high incidence and frequency in males at 100 mg/kg/day. There were no test item related effects on body weight, food consumption, ophthalmology, estrous cycle and clinical pathology parameters. At the end of the treatment period in the forestomach, adverse microscopic erosion/ulcer, inflammation/edema and squamous cell hyperplasia correlating with macroscopic white masses and/or gelatinous aspect
were observed at 100 mg/kg/day in both sexes. Similar non adverse microscopic changes of lower severities and incidence were noted at 30 mg/kg/day in males. These changes were reported with higher incidence and/or severity in males than in females. 

In conclusion, the oral administration of 1,4-butanediylbis[oxy(2-hydroxy-3,1-propanediyl) diacrylate in rats induced erosion/ulcer, inflammation/edema and secondary squamous cell hyperplasia in the forestomach from 30 mg/kg/day in males and at 100 mg/kg/day in females.
These changes were considered as adverse at 100 mg/kg/day in both sexes and contributed to the premature death of one male at 100 mg/kg/day.
Based on these adverse findings at 100 mg/kg/day, the No-Observed-Adverse-Effect Level (NOAEL) in this study was considered to be 30 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The study is considered as reliable with a klimisch score of 1.

System:

gastrointestinal tract

Organ:

stomach

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

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

90-day repeated toxicity study (CRL 2021)

The objective of this study was to evaluate the potential toxicity of the test item,
1,4-butanediylbis[oxy(2-hydroxy-3,1-propanediyl) diacrylate, following daily oral administration (gavage) to rats for 13 weeks (TG OECD 408).
Four groups of ten males and ten females Sprague-Dawley rats were treated daily by the oral route (gavage) with the test item, 1,4-butanediylbis[oxy(2-hydroxy-3,1-propanediyl) diacrylate, at dose levels of 0, 10, 30 or 100 mg/kg/day for 13 weeks (i.e. 91 or 92 days according to the necropsy schedule). The test item was administrated as an emulsion in the vehicle (corn oil) at a constant dosage volume of 5 mL/kg/day.
The following parameters and endpoints were evaluated in this study: survival, clinical signs, body weights, body weight gains, food consumption, ophthalmology, clinical pathology (hematology, coagulation, blood biochemistry, and urinalysis as well as thyroid hormones), estrous cycle, gross necropsy findings, organ weights, and histopathologic examinations.
The actual test item concentrations in the analyzed dose formulations were within an acceptable range of variations (-4.7% to +6.5%) when compared with the nominal values (nominal ± 15%).
The unscheduled death of one male at 100 mg/kg/day on Day 85 was attributed to the test item and considered to be related to adverse moderate erosion/ulceration and marked inflammation/edema in the forestomach.
Test item-related clinical signs in surviving animals consisted of minimal to severe
hypersalivation, observed at a low incidence and frequency in males and females at 30 mg/kg/day and females at 100 mg/kg/day, and at a high incidence and frequency in males at 100 mg/kg/day. There were no test item related effects on body weight, food consumption, ophthalmology, estrous cycle and clinical pathology parameters. At the end of the treatment period in the forestomach, adverse microscopic erosion/ulcer, inflammation/edema and squamous cell hyperplasia correlating with macroscopic white masses and/or gelatinous aspect
were observed at 100 mg/kg/day in both sexes. Similar non adverse microscopic changes of lower severities and incidence were noted at 30 mg/kg/day in males. These changes were reported with higher incidence and/or severity in males than in females. 

In conclusion, the oral administration of 1,4-butanediylbis[oxy(2-hydroxy-3,1-propanediyl) diacrylate in rats induced erosion/ulcer, inflammation/edema and secondary squamous cell hyperplasia in the forestomach from 30 mg/kg/day in males and at 100 mg/kg/day in females.
These changes were considered as adverse at 100 mg/kg/day in both sexes and contributed to the premature death of one male at 100 mg/kg/day.
Based on these adverse findings at 100 mg/kg/day, the No-Observed-Adverse-Effect Level (NOAEL) in this study was considered to be 30 mg/kg/day.

Combined repeated dose and reproduction / developmental screening (2013)

Test substance was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 50 mg/kg bw/d (test group 1), 150 mg/kg bw/d (test group 2) and 500 mg/kg bw/d (test group 3). Because of clinical findings and the premature death of 3 male animals in test group 3, the dose was reduced from 500 to 300 mg/kg bw/d from study day 6 onwards.

The objective of the study was to detect possible effects of the test substance on the integrity and performance of male and female reproductive systems including gonadal function, mating behavior, conception, gestation and parturition. Furthermore, it was intended to obtain information about the general toxicological profile including target organs and the no observed adverse effect level (NOAEL) after repeated oral administration. The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice.

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear.

Note: As a result of the premature death in 3 male animals of test group 3 (500 and 300 mg/kg bw/d) 3 male animal were mated in a 1:2 ratio, i.e. male animal No. 31 was mated with female animal Nos. 131 and 132, male No. 34 with female Nos. 133 and 134 and male animal No. 39 with Nos. 139 and 140. As a result of the premature death of male animal No. 39, female animal No. 140 was mated with male animal No. 36 from study day 16 onwards.

A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4. Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating and mating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, after the day of parturition (postnatal day [PND] 0) and on PND 4.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed under isoflurane anesthesia with CO2 and examined macroscopically for external and visceral findings.

Clinicochemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group towards the end of the administration period. Towards the end of the administration period a functional observational battery was performed and motor activity was measured in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group.

All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

 

In the high dose group, six male animals and 2 female animals were found dead during the study; in addition, 1 female animal was sacrificed moribund on gestation day 27. Respiration sounds were observed in 5 male and 3 female on several days during the study, labored respiration was observed in 2 male animals and 1 female animal on few study days and gasping respiration was observed in 1 male animal on day 6 of the postmating phase and in 1 female animal on day 20 of the gestation phase.

Piloerection were observed in 2 male animals and 2 female animals on several days during the study. Encrusted left eye was observed in 1 male animal on post-mating day 6. Blood in bedding and inability to deliver was seen in 1 female animal during the gestation phase from day 25 to day 27, light-brown vaginal discharge was observed on gestation day 27. Mean body weight was lower in male animals on post-mating day 7 (-8.6%). Mean body weight change values were decreased in male animals during the premating days 0-7 (-85.3%) and 0-13 (-42.1%). Increased absolute and relative neutrophil counts in males and decreased relative lymphocyte counts in males were observed. At pathology : mean terminal body weight was lower in male animals, i.e. -8%. Erosion/ ulcer and/or related squamous hyperplasia or inflammation/edema in the forestomach were observed in 9 of 10 male and all female animals. Erosions or ulcers in the glandular stomach were observed in 3 of 10 males.

 

In the intermediate group, respiration sounds were observed in 3 male and 2 female animals on several days during the study, labored respiration was observed in 3 male animals on few study days during the mating phase. Piloerection were in 1 male animal on day 8 and 9 of the mating phase and in 1 female animal on day 26 of the gestation phase. Mean body weight was significantly lower in male animals on mating day 8 (-4.3%). No test substance-related adverse findings were observed in clinical pathology. Erosion/ ulcer and/or related squamous hyperplasia or inflammation/edema in the forestomach were observed in 9 of 10 male and in 7 of 10 female animals.

Erosions or ulcers in the glandular stomach were observed in 1 of 10 female animals.

 

In th low dose group, only erosion/ ulcer and/or related squamous hyperplasia or inflammation/edema in the forestomach were observed in 6 of 10 male and 1 of 10 female animals.

 

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration by gavage of test substance to male and female Wistar rats revealed local pathological findings in the stomach at a dose level of 50 mg/kg bw/d and above. This finding was related to the irritating potential of the test substance.

Signs of systemic toxicity were observed at a dose level of 150 mg/kg bw/d (impaired body weight data in male animals) and above (premature deaths in both sexes), which were assumed to be related to the test substance administration.

Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 50 mg/kg bw/d for male animals and 150 mg/kg bw/d for female animals. With regard to effects in the stomach the NOAEL for local effects could not be set.

Justification for classification or non-classification

No organ damage or changes in clinical chemistry, urine or hematological parameters were observed. Reduced body weight and premature deaths were most likely the result of excessive local irritation and not of systemic toxicity. Even following the precautionary principle and assuming that some systemic toxicity occured in parallel to local irritation, no target organ was identified (with the only effect being reduced body weight) and the LOAEL (150 mg/kg) do not justify classification for single target organ toxicity after repeated exposure according to the Regulation EC n°1272/2008.