4-hydroxybutyl acrylate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02-16-2021 to 20-07-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

The duration of treatment covered a 14 weeks in-life period in the males including premat-ing, mating (mating pairs were from the same test group) and postmating period; a 10-weeks premating period, 3 days mating period, the entire gestation and approximately 4 weeks of lactation period in the females, approximately 3 weeks in-life period in the male rearing ani-mals and 8 days in-life period in the female rearing animals. In addition groups of 10 male and 10 female Wistar rats (F0 parental animals) and groups of 10 male and 10 female rearing animals, were treated with the test substance by gavage at doses of 12, 40 and 120 mg/kg body weight/day (mg/kg bw/d).

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-hydroxybutyl acrylate

EC Number:

219-606-3

EC Name:

4-hydroxybutyl acrylate

Cas Number:

2478-10-6

Molecular formula:

C7H12O3

IUPAC Name:

4-hydroxybutyl acrylate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: about 36+/+ 1 days
- Weight at study initiation: weight variation did not exceed 20 percent of the mean weight
- Housing: During the pretreatment, premating, mating and postmating period of the study, the rats were housed together (up to 5 (pretreatment) and 2 animals (premating) per sex and cage and 2 animals (males only during mating and postmating) per cage, respectively) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECNIPLAST, Hohenpeißenberg, Germany. During the mating, gestation and lactation period and after weaning the female rats were housed individually in Polycarbonate cages type III supplied by TECNIPLAST, Hohen-peißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany, with the following ex-ceptions: During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III. Pregnant animals and their litters were housed together until PND 21 in Polycar-bonate cages type III.
- Diet (e.g. ad libitum): ad libitum (Mouse and rat maintenance diet "GLP", Granovit AG, Switzerland)
- Water (e.g. ad libitum): ad libitum (drinking water)
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY:
The food used in the study will be assayed for chemical and microbial contaminants. Fed. Reg. Vol. 44, No. 91 (09 May 1979), p 27354 (EPA), will serve as the guideline for maximum tolerable contaminants. Additionally the levels of phytoestrogens should not exceed 350 µg of genistein equivalents/gram. According to recommendations of the GV-SOLAS, the total amount of bacteria must not exceed 1*105 per g food.
Drinking water analysis: The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring Department of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation will serve as the guideline for maximum tolerable contaminants

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral administration of a test substance has been proved useful worldwide in numerous studies for discovering a potential toxicological profile.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the test substance preparation, the specific amount of test substance will be weighed, topped up with drinking water in a graduated flask and intensely mixed with a magnetic stirrer until it is completely dissolved.
The test substance preparations will be prepared at intervals which guarantee that the test substance concentrations in the vehicle will remain stable.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in drinking water for a period of 7 days at room temperature was verified before the start of the study in a similar batch. At the beginning of administration, towards the middle and towards the end of administration each 1 sample was taken from the low, mid and high concentration for a concentration con-trol analysis. All test samples, plus a duplicate set of reserve samples, were withdrawn by staff of the Laboratory Reproduction Toxicology.
The reserve samples were stored at the Laboratory Reproduction Toxicology frozen (at -20 °C) Analysis of these samples were performed in case of equivocal analytical results with the original samples or after loss of/damage to original samples after agreement by the Study Director.

Duration of treatment / exposure:

The duration of treatment covered a 14 weeks in-life period in the males including premat-ing, mating (mating pairs were from the same test group) and postmating period; a 10-weeks premating period, 3 days mating period, the entire gestation and approximately 4 weeks of lactation period in the females, approximately 3 weeks in-life period in the male rearing ani-mals and 8 days in-life period in the female rearing animals.

Frequency of treatment:

once daily, 7 days per week (exception: no administration to animals being in labor)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

12 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

40 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

120 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on dose-range-finding studies

Positive control:

No positive control conducted.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- A cageside examination was conducted at least daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and between 2 and 5 hours after the administration. Abnormalities and changes were documented daily for each animal. Individual data of daily observations can be found in the raw data. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Detailed clinical observations (DCO) were performed in all animals once prior to the first ad-ministration (day 0) and at weekly intervals during the administration period. The examinations started in the morning.
- Detailed clinical observations checked in table No. 1 were included.

BODY WEIGHT: Yes
- body weight of the male and female parental animals and F1 rearing animals was deter-mined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results. During the mating period the parental females were weighed on the day of positive evi-dence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 0) and on PND 1, 4, 7, 10, 13, 17 and 21. Females without positive evidence of sperm, without litter and females after weaning (PND 21) were weighed weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- food consumption was determined once a week for male and female parental animals and F1 rearing animals, with the following exceptions: Food consumption was not determined after the 10nd premating week (male parental an-imals) and during the mating period (male and female parental animals). Food consumption of the females with evidence of sperm was determined on GD 0-7, 7-14 and 14-20. Food consumption of the females which gave birth to a litter was determined on PND 1-4, 4-7, 7-10, 10-13, 13-17 and 17-21. Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

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: Yes / No / Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- water consumption was determined once a week (over a period of 3 days) for the male and female parental animals and F1 rearing animals, with the following exceptions: Water consumption was not determined after the 10nd premating week (male parental animals) and during the mating period (male and female parental animals); Water consumption of the females with evidence of sperm was determined for GD 0-1, 7-8, 13-14 and 19-20; Water consumption of the females, which gave birth to a litter was determined for PND 1-2, 3-4, 6-7, 9-10, 12-13, 16-17 and 20-21.
Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Blood samples from all dams at PND 14 and all F0 males at termination were taken by punc-turing the retrobulbar venous plexus under isoflurane anesthesia for hormone measure-ment.
- examinations were performed in 5 F0 animals per sex and group towards the end of the ad-ministration period
- parameters checked in table No. 5 were examined.

CLINICAL CHEMISTRY: Yes
- Blood samples from all dams at PND 14 and all F0 males at termination were taken by punc-turing the retrobulbar venous plexus under isoflurane anesthesia for hormone measure-ment.
- examinations were performed in 5 F0 animals per sex and group towards the end of the ad-ministration period
- parameters checked in table No. 6 were examined.

URINALYSIS: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
end of administration period
- Dose groups that were examined:
all, first 5 surviving male and the first 5 surviving female animals with litter per group (in order of delivery)
- Battery of functions tested are checked int table No. 2-4: home cage observeratin; open field observation, sensory-motoric test; motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The animals will be sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals will be necropsied and assessed by gross pathology.
Organ weights: Were determinded as listed in table No.7

HISTOPATHOLOGY: Yes
The organs or tissues will be fixed in 4% neutral buffered formaldehyde solution or in modified Davidson's solution as listed in table No. 8

Statistics:

Means and standard deviations were calculated. Further statisctis were performed as listed in table No. 9

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All high-and several mid-dose males showed salivation during the entire study. Salivation was also observed in all high-dose females during the premating, gestation and lactation period, in two high-dose females during the mating period and in some mid-dose females during premating, gestation and lactation periods. The temporary salivation was considered to be test substance-induced.
No other clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the test groups (01 - 03; 12, 40 and 120 mg/kg bw/d) during the study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no test substance-related mortalities in any of the groups.
One female animal of test group 02 (40 mg/kg bw/d) was found dead during blood sampling on the day of scheduled sacrifice.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights and body weight change of all male and all female parental animals in all test substance-treated groups were comparable to the concurrent control values during the entire study. Mean body weight change of the high-dose parental males was statistically significantly below the concurrent control values during in-life days 42 - 49 and in the high-dose females during PND 1 - 4 (about 32%, respectively). As these changes lack consistence they were considered to be incidental findings

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of all male and all female animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

Water consumption of all male and female animals of all test substance-treated groups was largely comparable to the concurrent control throughout the entire study. Water consumption of the high-dose females was statistically significantly below the concur-rent control values during GD 13 - 14 (about 16%). As this decrease was not consistent, this is considered to be an incidental finding.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

After three months of administration, in males of test group 3 (120 mg/kg bw/d) prothrombin time (Hepatoquick’s test, HQT) was significantly reduced. The values were below the historical control range (males, HQT 34.4-42.3 sec). Therefore, this alteration was regarded as treatment related and adverse.

In addition, in males of test group 3 mean corpuscular hemoglobin concentration (MCHC) was significantly decreased whereas absolute reticulocyte counts were significantly increased. The measured red blood cell parameters (i.e., red blood cell (RBC) counts, hemoglobin and hematocrit values) were not altered. Absolute reticulocyte counts were within the historical control range (males, absolute reticulocytes 116.2-162.9 Giga/L). Therefore, MCHC decrease and reticulocyte count increase in males of test group 3 were regarded as incidental and not treatment related.

In dams at lactational day 14 (LD14) in test group 2 (40 mg/kg bw/d) relative monocyte counts were significantly decreased, but this change was not dose dependent. Therefore, this change was regarded as incidental and not treatment related.

After four months of administration, in F0 females of test group 3 (120 mg/kg bw/d) red blood cell (RBC) counts were significantly decreased. The mean RBC counts was 5% lower compared to study controls. No other red blood cell parameter was changed. Therefore, this isolated alteration was regarded as maybe treatment related but non adverse (ECETOC Technical Report No. 85, 2002).

In females of test group 3 relative basophil counts were significantly increased, but the values were within the historical control range (females, relative basophils 0.1-0.7 %). In females of test group 1 (12 mg/kg bw/d) relative lymphocyte counts were significantly lower whereas relative monocyte counts were significantly higher compared to controls, but both changes were not dose dependent. Therefore, the changes in this chapter were regarded as incidental and not treatment related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

After three months of administration, in males of test group 3 (120 mg/kg bw/d) LDL-cholesterol levels were significantly decreased whereas inorganic phosphate levels were significantly increased. Both parameters were outside historical control ranges (males, LDL-cholesterol, 0.17-0.27 mmol/L, inorganic phosphate 1.42-1.79 mmol/L). Therefore, these alterations were regarded as treatment related and adverse.

After four months of administration, in females of test group 3 total cholesterol and HDL-cholesterol levels were significantly increased. Total cholesterol levels were within the historical control range, HDL-cholesterol values were marginal above this range (females, cholesterol 1.02-2.14 mmol/L, HDL-cholesterol 1.07-1.26 mmol/L). However, these changes were regarded if ever treatment related as non-adverse. Additionally, in females of test groups 1, 2 and 3 (12, 40 and 120 mg/kg bw/d) total bilirubin values were significantly higher compared to controls. However, all values were within the historical control range (females, total bilirubin 1.38-2.36 µmol/L). Therefore, this change was regarded as incidental and not treatment related.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related changes among urinalysis parameters were observed in F0 males and females.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Home cage observations: No test substance-related or spontaneous findings were observed in male and female ani-mals of all test groups during the home cage observation.
Open field observations: The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.
Sensorimotor tests/reflexes: There were no test substance-related findings in male and female animals of all test groups.
Quantitative Parameters: No test substance-related impaired parameters were observed in male and female animals of all test groups. The statistically significantly lower value of landing foot-splay test in males of test group 02 was considered as spontaneous in nature and not treatment-related, as there was no dose-response.
Motor activity measurement: No treatment-related, adverse changes on motor activity data (summation of all intervals) was observed in the male and female animals of all test substance-treated groups in com-parison to the concurrent control values. The statistically significantly decreased number of beam interrupts in the low-dose males during interval 8 was considered to be spontaneous in nature and not treatment related since there was no relation to dose.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The significantly increased absolute and relative liver weight in males and the significantly increased relative liver weight in females in test group 03 (120 mg/kg bw/day) were regarded to be treatment-related. The still significantly increased relative liver weight of test group 02 males (40 mg/kg bw/day) were inside historical control values and therefore not regarded to be adverse.
In females of test group 02 and 03 (40 and 120 mg/kg bw/day) the reduced absolute adrenal gland weights were regarded to be unrelated to treatment as no dose-response relationship was present nor the relative organ weights were significantly altered, and the organ weights were within historical control values.
The relative increase of relative kidney weight in females of test group 03 (120 mg/kg bw/day) were minimally outside historical control values but were regarded to be unrelated to treatment as no histopathologic finding was observed that could explain the weight increase.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 120 mg/kg bw d the foci and the thickened margo plicatus in the forestomach were regarded to be treatment-related.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Forestomach:
In the forestomach, two females of test group 03 (120 mg/kg bw/day) showed erosion/ulcer. One male animal of the same test group revealed a moderate edema and severe focal hyperplasia of the squamous epithelium. This is a well-known secondary finding seen with erosion/ulcer. Therefore, in this animal an erosion/ulcer is most likely, although not observed on the slide investigated.
Four male and four female animals revealed focal hyperplasia of the margo plicatus. All these above-mentioned findings were regarded to be treatment-related.

Liver:
In the periportal area of the liver of males of test group 03 (120 mg/kg bw/day) microvesicular vacuolation of the hepatocytes was observed. A special stain for neutral fat (Oil Red O stain) was performed and showed a positive result.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.


The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls. In high dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals.
 

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormones:
After three months of administration in males of test group 3 (120 mg/kg bw/d) T4 values were significantly increased. The mean value was also above the historical control range (males, T4 44.65-73.22 nmol/L). T3 values and TSH values were not altered and neither any weight change nor any histopathologic finding was observed among these individuals. Therefore, the isolated increase of T4 values of males in test group 3 was regarded as non-adverse if at all treatment related.

After four months of administration, in females of test groups 1, 2 and 3 (12, 40 and 120 mg/kg bw/d) T4 values were significantly higher compared to controls. However, all values were within the historical control range (females, T4 26.66 60.54 nmol/L). T3 and TSH values were not changed. Therefore, this alteration was regarded as incidental and not treatment related.

At PND13, in F1 pups of test groups 1, 2 and 3 (12, 40 and 120 mg/kg bw/d) no changes of serum T4 and TSH levels were observed.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Discussion:

In an OECD 422 study, the test substance 4 -Hydroxybutyl Acrylate was administered daily as an aqueous solution to groups of 10 male and 10 female Wistar rats (F0 parental animals) and groups of 10 male and 10 female rearing animals by gavage at doses of 12, 40 and 120 mg/kg body weight/day (mg/kg bw/d)to screen for potential systemic, reproductive and developmental toxicity.Control animals (10 male and 10 female Wistar rats (F0 parental animals and F1 rearing animals) were dosed daily with the vehicle only (drinking water). The duration of treatment covered a 14 weeks in-life period in the males including premating, mating (mating pairs were from the same test group) and postmating period; a 10-weeks premating period, 3 days mating period, the entire gestation and approximately 4 weeks of lactation period in the females, approximately 3 weeks in-life period in the male rearing animals and 8 days in-life period in the female rearing animals.

The stability of these preparations was demonstrated over a period of 7 days under ambient conditions. Analyses confirmed the overall accuracy of the prepared concentrations of the test substance in the vehicle. 

Regarding clinical examinations, no test substance related, adverse signs of systemic toxicity were observed up to a dose of 120 mg/ kg bw/d. 

Transient salivation during a short time period after gavage dosing was noted for nearly all high-dose and some mid-dose male and female animals (F0 parents and F1 offspring) during all sections of the study. It is likely, that this temporary finding was induced by a local affection of the upper digestive tract, considering the evident histopathological signs of irritation found in the forestomach and the transition zone between fore- and glandular stomach of some high-dose animals. Salivation is not an adverse effect by itself, but at least for the high-dose group it can be considered as an outward sign reflecting adverse pathological findings in the stomach.

Neither water and food consumption nor body weight/body weight gain were adversely affected in any of the tested dose groups. 

Regarding clinical pathology, in males of test group 3 (120 mg/kg bw/d) prothrombin time was reduced which was most probably due to an increased synthesis of coagulation factors by liver cells. Additional lower LDL-cholesterol values in males of this test group may be also due to an increased uptake of LDL-cholesterol in the liver cells.

Regarding pathology, the forestomach and the liver were target organs. In the forestomach, one male and two females, all of test group 3 (120 mg/kg bw/day), revealed erosion/ulcer which corresponded to the macroscopic observation “focus”. The margo plicatus in the forestomach was macroscopically thickened in all males and four females of test group 3 (120 mg/kg bw/day). This was correlated to the histopathologic observation “hyperplasia, squamous epithelium”. Both findings were regarded to be treatment-related and locally adverse caused by the irritating effect of the test substance. 

The liver weight increase in males and females of test group 3 (120 mg/kg bw/day) were both regarded to be treatment-related. Males of this test group revealed an increase of periportal vacuolation of hepatocytes. These vacuoles were positive (neutral fat) in the ORO stain. Therefore, the liver findings in males of test group 3 (120 mg/kg bw/day) were in combination with findings in clinical pathology regarded to be treatment-related and adverse.

In females, there was no histopathologic finding that could explain the weight increase. As no adverse findings in females of test group 3(120 mg/kg bw/day) in clinical pathology were observed, the weight increase of the liver was assessed to be not adverse. 

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Regarding fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups during the entire study. All F0 parental animals proved to be fertile. Mating behavior, conception, implantation and parturition were not affected. 

Regarding developmental toxicity, no signs of developmental toxicity were noted in any of the treated groups. Pup status, viability, survival and growth, as well as timing of puberty showed no treatment-related, adverse findings.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present OECD 422 combined repeated dose tox-icity study with the reproductive/developmental screening test in Wistar rats the oral admin-istration of 4-Hydroxybutyl Acrylate by gavage to male and female Wistar rats resulted in signs of systemic toxicity at a dose of 120 mg/kg bw/d, considering clinical pathological and pathological findings. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was the mid dose of 40 mg/kg bw/d for male and female Wistar rats.