4-hydroxybutyl acrylate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Data for 4 -hydroxybutyl acrylate.

- NOAEL(fertility and reproductive performance / developmental toxicity): 120 mg/kg bw d (rat, OECD 408/422)

- NOAEL (maternal toxicity): 40 mg/kg bw d

4 -hydroxybutyl acrylate is not considered to be reproductive toxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please see for more information the read-across justification in Section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOEC

Remarks:

reproductive toxicity

Effect level:

ca. 0.269 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 75 ppm; the highest concentration tested.

Remarks on result:

other: Result read-across CAS No. 96-33-3

Remarks:

Correction for molecular weight not required.

Dose descriptor:

NOEC

Remarks:

parental local toxicity

Effect level:

ca. 0.019 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 5 ppm; based on histologic changes in nasal tissues.

Remarks on result:

other: Result read-across CAS No. 96-33-3

Remarks:

Correction for molecular weight not required.

Key result

Dose descriptor:

NOAEL

Effect level:

240 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: general toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

460 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fertility

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no reproductive effects observed

Remarks on result:

other: Result read-across CAS No. 141-32-2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Remarks on result:

other: Reasult read-across CAS No. 141-32-2

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: local effects (nonglandular stomach)

Remarks on result:

other: Reasult read-across CAS No. 141-32-2

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Remarks on result:

other: Result read-across CAS No. 25584-83-2

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed

Remarks on result:

other: Result read-across CAS No. 25584-83-2

Dose descriptor:

NOAEL

Remarks:

general, systemic toxicity

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Remarks on result:

other: Result read-across CAS No. 25584-83-2

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: irritation in forestomach and duodenum

Remarks on result:

other: Result read-across CAS No. 25584-83-2

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOEC

Remarks:

reproductive toxicity

Effect level:

ca. 0.269 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 75 ppm; the highest concentration tested.

Remarks on result:

other: Result read-across CAS No. 96-33-3

Remarks:

Correction for molecular weight not required.

Dose descriptor:

NOEC

Remarks:

parental local toxicity

Effect level:

0.019 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 5 ppm; based on histologic changes in nasal tissues.

Remarks on result:

other: Result read-across CAS No. 96-33-3

Remarks:

Correction for molecular weight not required.

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOEC

Remarks:

developmental toxicity

Generation:

F1

Effect level:

ca. 0.092 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 25 ppm; based on decreases in pup body weights at 75 ppm which were secondary to parental toxicity.

Remarks on result:

other: Result read-across CAS No. 96-33-3

Remarks:

Correction for molecular weight not necessary.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Remarks on result:

other: Result read-across CAS No. 141-32-2

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: local effects (nonglandular stomach)

Remarks on result:

other: Result read-across CAS No. 141-32-2

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Remarks on result:

other: Result read-across CAS No. 25584-83-2

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOEC

Remarks:

developmental toxicity

Generation:

F2

Effect level:

ca. 0.092 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 25 ppm; based on decreases in pup at 75 ppm which were secondary to parental toxicity.

Remarks on result:

other: Result read-across CAS No. 96-33-3

Remarks:

Correction for molecular weight not necessary.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

0.269 mg/L air (analytical)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

no

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: OECD guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

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

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 se-lected 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

Route of administration:

oral: gavage

Vehicle:

water

Details on 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.

Details on mating procedure:

- M/F ratio per cage:1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: Vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted " gestation day (GD) 0" and the following day "gestation day (GD) 1"

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 La-boratory 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)

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

12 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

120 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals
10 F1 rearing 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.

Parental animals: Observations and examinations:

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

Oestrous cyclicity (parental animals):

For a minimum of 3 weeks prior to mating estrous cycle length was evaluated by daily analy-sis of vaginal smear for all F0 female parental rats. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, sperm motility, sperm morphology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups,

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead]

OTHER: On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood were sampled for determination of thyroid hormone con-centrations. All pups were examined externally and eviscerated; their organs were assessed macroscopically.

Postmortem examinations (parental animals):

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

Postmortem examinations (offspring):

SACRIFICE, GROSS NECROPSY and HISTOPATHOLOGY / ORGAN WEIGTHS:
On PND 4, as a result of standardization, the surplus pups or 2 preferably female pups per litter, respectively, were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations (see 3.9.). After sacrifice, the pups were examined externally and eviscerated, and the organs were assessed macroscop-ically.
On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone con-centrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formalde-hyde solution and were transferred to the Pathology Laboratory for possible further pro-cessing. The remaining bodies were necropsied as far as possible.
On PND 21, the surplus F1 generation pups that were not used as F1 rearing animals were likewise be sacrificed under isoflurane anesthesia by CO2. After sacrifice, these pups were examined externally, eviscerated and their organs were assessed macroscopically.
Pups showing clinical symptoms or gross-morphological findings might be further examined using appropriate methods. Organs/tissues with gross-morphological findings might be pre-served in a suitable manner for potential histopathological examination.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation.

Statistics:

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

Reproductive indices:

Male mating index, male fertility index, female mating index, female fertility index, gestation index, live birth index, postimplantation index

Offspring viability indices:

Viability index, lactaion index, sex ration, anogenital index

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.

Dermal irritation (if dermal study):

not examined

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 (No. 127 - 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

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.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data, generated during the last 3 weeks prior to mating for the F1 litter, re-vealed regular cycles in the females of all test groups 00 - 03. The mean estrous cycle duration was similar: 4.0 / 4.1 / 4.0 and 4.0 days in test groups 00 - 03, respectively.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the cauda epididymidis no treatment-related effects were observed.
The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls.
In males of test group 3 (120 mg/kg bw/d) sperm head counts in testis were significantly lower compared to study controls. However, the values were within the historical control range (sperm head counts in testis 87-131 Mio/g testis). Therefore, this isolated alteration was regarded as incidental and not treatment related.

Reproductive performance:

no effects observed

Description (incidence and severity):

Male reproduction data:
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups (00 - 03). Fertility was proven for all F0 parental males within the scheduled mating interval for F1 litter.
Thus, the male fertility index was 100% in all test groups.
 
Female reproduction and delivery data:
The female mating index calculated after the mating period for F1 litter was 100% in all test groups (00 - 03). The mean duration until sperm was detected (GD 0) varied between 2.4 and 2.7 days with-out any relation to dosing.
All female rats delivered pups or had implants in utero:
The fertility index was 100% in all test groups.
The mean duration of gestation values varied between 22.1 / 22.1 / 22.1 and 22.3 in test groups 00 - 03, respectively.
The gestation index was 100% in all test groups 00 - 03.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (13.1 / 12.8 / 12.8 and 13.1 implants/dam in test groups 00 - 03, respectively). Furthermore, there were no indications for test substance-induced intrau-terine embryo-/fetolethality since the post-implantation loss did not show any significant dif-ferences between the groups (13.0 / 11.0 / 6.0 and 13.3 mean% in test groups 00 - 03, re-spectively), and the mean number of F1 pups delivered per dam remained unaffected (11.4 / 11.3 / 12.0 and 11.4 pups/dam in test groups 00 - 03, respectively).
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 97.4% / 100% / 97.5% and 98.2% in test groups 00 - 03, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups.
Thus, the test substance 4-Hydroxybutyl Acrylate did not adversely affect reproduction and delivery of the F0 generation parental females.

Dose descriptor:

NOAEL

Remarks:

repoductive

Effect level:

120 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: high dose

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- F1 pups
There were no test substance related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

- F1 rearing animals
Most of the high-dose males and 2 high-dose females showed salivation during their entire study. 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 animals in any of the test groups (11 - 13; 12, 40 and 120 mg/kg bw/d) during the study. No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

- F1 pups
The pup mortality based on total number of stillborn pups, dead pups, pups sacrificed moribund and cannibalized pups.
PND 0: 3 male in control goup, 2 male and 1 female in test group 2 (40 mg/kg) and 2 male in test group 3 (120 mg/kg)
PND 1-4: 2 female in test group 1 (12 mg/kg) and 1 female in test group 2 (40 mg/kg)
PND 8-14: 1 male in test group 3 (120 mg(kg)

- F1 rearing animals
There were no test substance-related or spontaneous mortalities in any of the groups.

The viability index indicating pup survival during lactation (PND 0 - 4) varied between 100%/ 98.6% / 99.3% and 100% in test groups 00 - 03, respectively.

The pups surviving index indicating pup survival during lactation (PND 4 - 21) varied be-tween 73.5%/ 75% / 75% and 73.8% in test groups 00 - 03, respectively. These figures factor in the scheduled sacrificed pups on PND 13 for blood sampling for hormone measurement.

Thus, the test substance did not influence pup survival in any of the treated groups (12, 40 and 120 mg/kg bw/d).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- F1 pups
The mean body weights and body weight change of all male and female pups in all test substance-treated groups were comparable to the concurrent control values throughout the entire study.
One male and one female runt was seen in the control, one male and three female runts were seen in test group 02 and one female runt was seen in test group 03.

- F1 rearing animals
Mean body weights and body weight change of all male and all female animals in all test substance-treated groups were comparable to the concurrent control values during their entire study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- F1 rearing animals
Food consumption of all male and all female animals of all test substance-treated groups was comparable to the concurrent control values throughout their entire study.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

- F1 rearing animals
Water consumption of all male and all female animals of all test substance-treated groups was comparable to the concurrent control values throughout their entire study.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

- F1 pups
The anogenital distance and anogenital index of all test substance treated male and female pups were comparable to the concurrent control values.
Vaginal opening
Each female F1 pup, which was selected to become a F1 rearing females, was evaluated for commencement of sexual maturity. The first day when vaginal opening was observed was PND 28, the last was PND 35. The mean number of days to reach the criterion in the control and 12, 40 and 120 mg/kg bw/d test groups amounted to 30.3 / 30.8 / 31.2 and 31.6 days, showing no significant differences. The mean body weight on the day, when vaginal open-ing was recorded, amounted to 98.2 g / 97.8 g / 96.5 g and 103.2 g in test groups 00-03.
Preputial separation
Each male F1 pup, which was selected to become a F1 rearing male, was evaluated for commencement of sexual maturity. The first day when preputial separation was observed was PND 39, the last was PND 47. The mean number of days to reach the criterion in the control and 12, 40 and 120 mg/kg bw/d test groups amounted to 42.0 / 43.1 / 42.9 and 42.8 days, showing no significant differences. The mean body weight on the day, when preputial separation was recorded, amounted to 187.7 g / 196.2 g / 190.7 g and 189.4 g in test groups 00-03.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

- F1 pups
The number and percentage of male pups showing nipple/ areolae anlagen was not influenced by the test substance when examined on PND 13. However, all animals are being habitually rechecked for nipples/areolae on PND 20, one day prior to weaning. During this re-examination no nipple/ areolae anlagen were detected in all male pups of test groups 01 and 03.
The single pup with nipple detected in test group 02 during re-examination on PND 20 was considered as spontaneous in nature and not treatment-related, as there was no dose-response and no nipple/ areolae anlagen were detected in the remaining pups of test groups 02.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- F1 pups
A few pups showed spontaneous findings at gross necropsy, such as diaphragmatic hernia and dilated renal pelvis. These findings occurred without any relation to dosing. Thus, all these findings were con-sidered not to be associated to the test substance.

- F1 rearing animals
A few F1 rearing animals showed the spontaneous finding dilated renal pelvis at gross necropsy. This finding occurred without any relation to dosing. Thus, this finding was considered not to be associated with the test substance.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio:
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 21 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
Pup number and status at delivery:
The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were evenly distributed about the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
Pup viability:
The viability index indicating pup survival during lactation (PND 0 - 4) varied between 100%/ 98.6% / 99.3% and 100% in test groups 00 - 03, respectively.
The pups surviving index indicating pup survival during lactation (PND 4 - 21) varied be-tween 73.5%/ 75% / 75% and 73.8% in test groups 00 - 03, respectively. These figures factor in the scheduled sacrificed pups on PND 13 for blood sampling for hormone measurement.
Thus, the test substance did not influence pup survival in any of the treated groups (12, 40 and 120 mg/kg bw/d).

Thyroid hormones
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.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not specified

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

120 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: high dose

Critical effects observed:

no

Reproductive effects observed:

no

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.

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.
The NOAEL for fertility and reproductive performance was 120 mg/kg bw/d for male and fe-male Wistar rats. The NOAEL for developmental toxicity was 120 mg/kg bw/d.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

120 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

269 mg/m³

Study duration:

subchronic

Species:

rat

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Data are available for 4 -hydroxybutyl acrylate and for the structurally analogous substances methyl acrylate (CAS No. 96-33-3) and butyl acrylate (CAS No. 141 -32 -2)

Data for 4 -HBA CAS No. 2478 -10 -6 (combined OECD 408/422 by oral gavage):

4-HBA 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). 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 dose selection was based on the results of 14 d range-finding studies (0; 50; 150; 200 and 600 mg/kg bw/d), the animals of the 600 mg/kg bw/d were sacrificed moribund / died prematurely, therefore, organ weights were not recorded. Significantly increased absolute and relative liver weights were described at 150 mg/kg bw/d and 200 mg/kg bw/d, dose-dependent adverse local effects to forestomach and stomach occurred at 150 mg/kg bw/d and higher.

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.

After 10 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 were detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females.

A detailed clinical observation (DCO) was performed in all F0 animals before the start of the administration period and, as a rule, thereafter at weekly intervals for the F0 animals and F1 rearing animals. Water consumption of the F0 parents during premating and F1 rearing animals was determined once a week (over a period of 3 days). However, during gestation and lactation water consumption of the F0 females were determined on gestation days (GD) 0-1, 7-8, 13-14, and 19-20 and on postnatal days (PND) 1-2, 3-4, 6-7, 9-10, 12-13, 16-17 and 20-21. Food consumption of the F0 parents during premating and F1 rearing animals was determined once weekly. In dams food consumption was determined for GD 0-7, 7-14, 14-20 and PND 1-4, 4-7, 7-10, 10-13, 13-17 and 17-21. In general, body weights of F0 parents and F1 rearing animals were determined once weekly. However, during gestation and lactation F0 females were weighed on GD 0, 7, 14 and 20 and on PND 0, 1, 4, 7, 10, 13, 17 and 21. Estrous cycle data were evaluated for F0 generation females over a three weeks period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1, 4, 7, 13 and 21. Their viability was recorded. At necropsy, all pups were sacrificed with CO₂under isoflurane anesthesia (except the selected pups for blood sampling) and examined macroscopically for external and visceral findings.

Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1.All surviving male pups were examined for the presence or absence of nipple/areola anlagen on PND 12. If nipple/areola anlagen were recorded, all surviving male pups were carefully re-examined one day prior to necropsy (PND 20).

Blood samples were taken from all surplus pups at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia for hormone measurement. Clinico-chemical and hematological examinations were performed in 5 F0 animals per sex and group towards the end of the administration period. Blood samples from all dams at PND 14 and all F0 males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement. Urine samples for clinical pathological investigations were withdrawn from all F0 parents. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group. Various sperm parameters (motility, sperm head count, morphology) were assessed in the male F0 parental animals at scheduled sacrifice or after appropriate staining. 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.

 

The following test substance-related relevant effects/findings were noted in the F0 parental animals at 120 mg/kg bw/d:

• Salivation after gavage dosing in all male animals during the entire study and in all female animals during premating, gestation and lactation period, and in 2 females during mating period
• Reduced prothrombin time in F0 males
• Decreased LDL-cholesterol in F0 males
• Increased inorganic phosphate values in males
• signs of erosion/ulcer in the forestomach one male and two females
• focal hyperplasia of the margo plicatus of four males and four females
• vacuolation of the hepatocytes in the periportal area of the liver of males

 

No test substance-related, adverse findings (also including macroscopically for external and visceral findings) were observed at F1 pups and rearing animals at 120 mg/kg bw/d. No test substance-related, adverse findings were observed in the F0 animals and F1 pups and rearing animals at 15 and 40 mg/kg bw/d.

 

In conclusion, under the conditions of the present OECD 408/422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the oral administration of 4HBA 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. The NOAEL for fertility and reproductive performance was 120 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was 120 mg/kg bw/d (BASF, 2022)

Data from the structural analogue Methyl acrylate (MA, CAS No. 96-33-3):

In a two-generation study according to OECD TG 416 (Dow Chemical Co., 2009) groups of 27 male and female Crl:CD(SD) rats were whole-body exposed to the structural analogue methyl acrylate vapours at target concentrations of 0, 5, 25, and 75 ppm for six hours/day, seven days/week, resulting in actual average concentrations of 0, 5.3 ± 0.2, 25.7 ± 0.3, and 75.4 ± 0.6 ppm, respectively (corresponding to approx. 0, 0.019, 0.092, and 0.269 mg/L).Rats were exposed daily for approximately ten weeks prior to breeding, and continuing through breeding, gestation and lactation for two generations. Maternal rats were not exposed after GD 20 through LD 4 in order to allow for parturition and initiation of lactation. Exposure of maternal rats continued from LD 5 – LD 28. In-life parameters included clinical observations, feed consumption, body weights, estrous cyclicity, reproductive performance, pup survival, pup body weights, and puberty onset.In addition, post-mortem evaluations included gross pathology, histopathology, organ weights, oocyte quantitation and sperm count, motility and morphology in adults, and gross pathology and organ weights in weanlings. Treatment-related effects in parental rats exposed to 75 ppm included decreased body weight and feed consumption in males and females throughout most of the two generation study. There were no effects on body weight or feed consumption at 25 or 5 ppm. Treatment-related, adverse histopathologic effects were present in the nasal tissues of P1 and P2 males and females exposed to 25 or 75 ppm. The incidence and severity of the nasal effects were concentration dependent. Degeneration with regeneration of the olfactory epithelium (very slight to moderate) occurred in all P1 and P2 males and females exposed to 75 ppm. Very slight olfactory epithelial degeneration, without accompanying regenerative hyperplasia, was noted in some of the P1 and P2 females and P2 males exposed to 25 ppm. There were several histopathologic effects that accompanied the degeneration of the olfactory epithelium. Very slight or slight degeneration of the olfactory nerve was present in most of the P1 and P2 males and females exposed to 75 ppm, and one P1 male exposed to 25 ppm. Very slight or slight chronic-active inflammation was present in 16/27 P1 males, 20/27 P1 females, 14/27 P2 males, and 8/27 P2 females exposed to 75 ppm, and in one or two males and females from both generations exposed to 25 ppm. Very slight necrosis of individual olfactory epithelial cells was present in most of the P1 and P2 males and females exposed to 75 ppm, and a few P1 and P2 animals (one to four per sex) exposed to 25 ppm. Very slight mineralization of the olfactory epithelium was present in one or two P1 and P2 animals exposed to 25 ppm, and in 6/27 P1 males, 4/27 P1 females, 16/27 P2 males and 14/27 P2 females exposed to 75 ppm. Other nasal effects consisted of an increase in the incidence of very slight or slight hyperplasia of the transitional epithelium in P1 and P2 males and females exposed to 25 or 75 ppm, and an increase in the incidence of very slight or slight hyperplasia and hypertrophy of the respiratory epithelium in P1 males and females exposed to 25 or 75 ppm, and in P2 males and females exposed to 75 ppm. There were no treatment-related histopathologic effects in P1 or P2 animals exposed to 5 ppm.

No treatment-related effects were seen in reproductive function or pup survival. However, pup body weights of the 75 ppm exposure group were decreased on postnatal day 14-28 in both generations. There were no effects on pup body weight in rats exposed to 25 or 5 ppm. The effects on pup body weight, as well as the changes in parental body weight and feed consumption, likely were secondary changes all stemming from nasal irritation and resultant stress.

In summary, the no-observed-effect concentration (NOAEC) for parental systemic toxicity was determined to be 5 ppm (= ca. 0.019 mg/L) and was based on histologic changes in the nasal tissues seen at higher concentrations.The NOAEC for developmental toxicity was 25 ppm (= ca. 0.092 mg/L), based on decreases in pup body weight at 75 ppm which were secondary to parental toxicity. The NOAEC for reproductive toxicity was 75 ppm (= ca. 0.269 mg/L), the highest concentration tested.  

Conclusion

In a 2-generation-study with the structural analogue methyl acrylate by the inhalation route, no effects on reproductive function (i.e. fertility) were observed. Based on the structural similarities between the substances, it can be safely assumed that 4-hydroxybutyl acrylate does not cause any toxicity to reproduction.  

Data from the structural analogue n-butyl acrylate (n-BA, CAS No. 141-32-2):

For n-butyl acrylate an extended one generation study according to OECD 443 and GLP was performed. 30 Crl:CD(SD) rats were exposed to 20, 50 and 150 mg/kg bw/day by oral (gavage) exposure route (Acrylate Reach TF, 2017).

There were no test substance-related effects on survival for F0 and F1 animals at any dosage level. No test substance-related clinical observations were noted for F0 and F1 animals at any dosage level. Mean body weights, body weight gains, food consumption, and food efficiency in the 20, 50, and 150 mg/kg/day F0 and F1 males and females were unaffected by test substance administration. No test substance-related effects were noted on F0 reproductive performance (male and female mating and fertility, male copulation, and female conception indices), the mean number of days between pairing and coitus, mean gestation lengths, or the process of parturition. In addition, there were no test substance-related effects on F0 or F1 estrous cyclicity or spermatogenic parameters (testicular and epididymal sperm concentrations, sperm production rate, sperm motility, and sperm morphology) at any dosage level. There were no test substance-related effects on the number of F1 pups born, live litter size, percentage of males at birth, F1 postnatal survival, clinical observations, anogenital distance, offspring body weights, necropsy findings, or developmental landmarks (areolae/nipple retention, vaginal patency, and balanopreputial separation). No test substance-related effects on clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were noted for F0 and F1 animals at any dosage level. In addition, no test substance-related effects on serum levels of T4 (thyroxine) or TSH (thyroid stimulating hormone) were noted in F0 and F1 males or females or F1 pups (on PND 4 and 21). Test substance-related histologic changes were observed in all dosage groups in the F0 generation and F1 males and females in Cohort 1A. Epithelial hyperplasia and/or hyperkeratosis was observed in the nonglandular stomach in all test substance-treated groups examined. Mild to moderate changes in the 150 mg/kg/day group males and females of the F0 and F1 generations were considered adverse in this study. Microscopic changes in the stomach were associated with the gross observation of thickened nonglandular stomach, but were not associated with any clinical pathology, organ, or body weight changes. In the F0 generation, a nonadverse increased incidence of biliary hyperplasia (males and females) and random hepatocellular necrosis (males) were observed in the liver in the 150 mg/kg/day group. Additionally, nonadverse test substance-related microscopic findings (increased severity of mineralization at the corticomedullary junction) were observed in the kidneys of the 150 mg/kg/day group F0 females. Thickened stomachs were noted in the 50 and 150 mg/kg/day group F1 males and in the 150 mg/kg/day group F1 females at the scheduled necropsies for Cohort 1B; this finding was considered test substance-related and adverse in the 150 mg/kg/day group males and females. No other test substance-related internal findings were observed at any dosage level for F1 Cohort 1B animals. No test substance-related effects on the mean number of F0 implantation sites or number of unaccounted-for sites were noted at any dosage level. No test substance-related macroscopic findings were observed in F1 pups that were found dead, culled on PND 4, or examined at the scheduled necropsy on PND 21; F1 pup organ weights on PND 21 were unaffected by test substance administration. No test substance-related effects on ovarian primordial follicle counts were noted in the F0 females suspected of reduced fertility or F1 Cohort 1A females. There were no test substance-related effects on organ weights noted for F0 and F1 males and females at any dosage level. Due to the absence of systemic toxicity noted for F0 and F1 males and females throughout the study, a dosage level of 150 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 and F1 male and female systemic toxicity when n-butyl acrylate was administered orally by gavage to Crl:CD(SD) rats. Epithelial hyperplasia and/or hyperkeratosis in the nonglandular stomach noted in the 150 mg/kg/day group F0 and F1 males and females were considered adverse; based on these results, 50 mg/kg/day was considered to be the NOAEL and 150 mg/kg/day was considered to be the lowest-observed-adverse-effect level (LOAEL) for local effects in the F0 and F1 generations. Based on the lack of effects noted for F1 litters, a dosage level of 150 mg/kg/day was considered to be the NOAEL for neonatal toxicity. There was no evidence of reproductive toxicity at any dosage level based on evaluation of reproductive performance in the F0 generation and sperm measurements and estrous cyclicity in the F0 and F1 generations. Therefore, the NOAEL for F0 and F1 reproductive toxicity was considered to be 150 mg/kg/day.

Data from the structural analogue Hydroxypropyl acrylate (HPA, CAS No. 25584-83-2):

Hydroxypropylacrylate was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 15, 50 and 150 mg/kg body weight/day (mg/kg bw/d). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (drinking water). The duration of treatment covered a 2-week premating and a mating period for both sexes, approximately 2 days post-mating in males, as well as gestation and lactation in females.

The following adverse treatment-related findings were noted at 150 mg/kg bw/d. Decreased food consumption in the females during the entire premating period (up to 7% below control). Minimal to slight thickening of the mucosa of the duodenum correlating to the macroscopically observed dilation in all male and 9/10 females. Focal hyperplasia of the duodenal mucosa in 1/10 female animals. Diffuse squamous hyperplasia of the forestomach in all male animals (graded slight or moderate) and in 7/10 female animals (graded minimal or moderate). Erosion/ulcer in the cranial part of the forestomach in 6/10 male and 2/10 female animals. At 50 mg/kg bw/d minimal thickening of the wall of the duodenum correlating to the macroscopically observed dilation in 2/10 male animals. Minimal diffuse squamous hyperplasia of the forestomach in 6/10 male and female animals. At 15 mg/kg bw/d no test substance-related adverse findings were observed. No test substance-related adverse findings were observed for the F1 generation.

Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the NOAEL (no observed adverse effect level) for general, systemic toxicity of Hydroxypropylacrylate was 150 mg/kg bw/d for male and female rats. Based on pathological findings characteristic of irritation in forestomach and duodenum in F0 parental rats of both sexes at 150 and 50 mg/kg as well as corresponding temporary reductions of food consumption in F0 females at 150 mg/kg bw/d a NOAEL of 15 mg/kg bw/d was determined for local effects in the gastrointestinal tract.

Data from the structural analogue Hydroxyethyl acrylate (HEA, CAS No.818 -61 -1:

A study according to OECD TG 422 for HEA is available(HARTF 2020b).

In this study HEA was administered daily to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 12, 40 and 120 mg/kg body weight/day (mg/kg bw/day, test groups 1-3, respectively) to screen for potential systemic, reproductive and developmental toxicity the substance administration resulted in signs of parental toxicity at the mid- and high-dose of 40 and 120 mg/kg bw/day, such as a combination of clinical signs and gastrointestinal pathology, being the consequence of the irritating properties of the test item. Thus, the NOAEL for general systemic toxicity was 40 mg/kg bw/day for male and female Wistar rats based on the reduced food consuption (lactation phase) and the significant increased liver weights and the NOAEL for local effects (gastrointestinal pathology) was 12 mg/kg bw/day. The NOAEL for reproductive performance and fertility was set to 120 mg/kg bw/day for male and female Wistar rats. The NOAEL for developmental toxicity was 120 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

Data for 4 -hydroxybutyl acrylate.

- NOAEL( fertility and reproductive performance / developmental toxicity): 120 mg/kg bw d (rat, OECD 408/422

- NOAEL (maternal toxicity): 40 mg/kg bw d

Data from the structural analogue 2-hydroxyethyl acrylate (CAS No. 818-61-1)

(New Zealand rabbits, OECD TG 414, oral)

- NOAEL (maternal toxicity) = 20 mg/kg bw/d

- NOAEL (developmental toxicity) = 60 mg/kg bw/d (no adverse effects observed)

(Sprague-Dawley rats, no guideline, vapour inhalation):

- NOAEC (maternal toxicity) = 5 ppm (= 0.0241 mg/L),

- NOAEC (developmental toxicity) = 10 ppm (= 0.0482 mg/L)

Data from the structural analogue hydroxypropyl acrylate (CAS No. 25584-83-2)

(Sprague-Dawley rats, no guideline, vapour inhalation):

- NOAEC (maternal toxicity) = 1 ppm (= 0.0054 mg/L),

- NOAEC (developmental toxicity) = 10 ppm (= 0.054 mg/L).

Data from the structural analogue methyl acrylate (CAS No. 96-33-3) (Himalayan rabbits,OECD TG 414, vapour inhalation):

- NOAEC (maternal toxicity) = 15 ppm (= 0.0553 mg/L),

- NOAEC (developmental toxicity) = 45 ppm (= 0.1556 mg/L).

Data from the structural analogue n-butyl acrylate (CAS No. 141 -32 -2):

- NOAEL = 400 mg/kg bw (rabbits, OECD TG 414)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Please see for more information the read-across justification in Section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEC

Remarks:

systemic toxicity / rat

Effect level:

ca. 0.005 mg/L air (nominal)

Based on:

test mat.

Remarks:

corresponding to 1 ppm

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: Result read-across CAS No. 25584-83-2

Remarks:

Correction for molecular weight not necessary.

Dose descriptor:

NOAEC

Remarks:

systemic toxicity / rat

Effect level:

ca. 0.024 mg/L air (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: Result read-across CAS No. 818-61-1

Remarks:

Correction for molecular weight not necessary.

Key result

Dose descriptor:

NOAEC

Remarks:

rabbit

Effect level:

15 ppm

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: Result read-across CAS No. 96-33-3

Remarks:

Correction for molecular weight not necessary.

Dose descriptor:

NOAEC

Remarks:

rat

Effect level:

ca. 0.089 mg/L air (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: Result read-across CAS No. 96-33-3

Dose descriptor:

NOAEL

Remarks:

rabbit

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal systemic toxicity & maternal developmental toxicity

Remarks on result:

other: Result read-across CAS No. 141-32-2

Dose descriptor:

LOAEC

Remarks:

rat

Effect level:

ca. 0.52 mg/L air (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: Result read-across CAS No. 141-32-2

Dose descriptor:

NOAEC

Remarks:

rat

Effect level:

ca. 0.13 mg/L air (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: Result read-across CAS No. 141-32-2

Dose descriptor:

NOAEL

Remarks:

mouse

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: Result read-across CAS No. 141-32-2

Dose descriptor:

NOAEL

Remarks:

mouse

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: Result read-across CAS No. 141-32-2

Key result

Abnormalities:

no effects observed

Key result

Dose descriptor:

NOAEC

Remarks:

rat

Effect level:

ca. 0.054 mg/L air (nominal)

Based on:

test mat.

Remarks:

corresponding to 10 ppm

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: Result read-across CAS No. 25584-83-2

Remarks:

Correction for molecular weight not necessary.

Dose descriptor:

NOAEC

Remarks:

rat

Effect level:

ca. 0.048 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: Result read-across CAS No. 818-61-1

Remarks:

Correction for molecular weight not necessary.

Key result

Dose descriptor:

NOAEC

Remarks:

rabbit

Effect level:

45 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: Result read-across CAS No. 96-33-3

Remarks:

Correction for molecular weight not necessary.

Dose descriptor:

NOAEC

Remarks:

rat

Effect level:

>= 0.357 mg/L air (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: teratogenicity

Remarks on result:

other: Result read-across CAS No. 96-33-3

Dose descriptor:

LOAEC

Remarks:

rat

Effect level:

ca. 0.357 mg/L air (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: fetotoxicity

Remarks on result:

other: Result read-across CAS No. 96-33-3

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Result read-across CAS No. 141-32-2

Dose descriptor:

NOAEC

Remarks:

rat

Effect level:

ca. 0.52 mg/L air (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: fetotoxicity

Remarks on result:

other: Result read-across CAS No. 141-32-2

Dose descriptor:

NOAEC

Remarks:

rat

Effect level:

ca. 1.57 mg/L air (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: teratogenicity

Remarks on result:

other: Result read-across CAS No. 141-32-2

Dose descriptor:

NOAEC

Remarks:

rat

Effect level:

ca. 0.13 mg/L air (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: embryotoxicity

Remarks on result:

other: Result read-across CAS No. 141-32-2

Dose descriptor:

NOAEC

Remarks:

rat

Effect level:

ca. 1.31 mg/L air (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: teratogenicity

Remarks on result:

other: Result read-across CAS No. 141-32-2

Dose descriptor:

NOAEL

Remarks:

mouse

Effect level:

2 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: teratogenicity

Remarks on result:

other: Result read-across CAS No. 141-32-2

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: OECD guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

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

Species:

rat

Strain:

Wistar

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

Route of administration:

oral: gavage

Vehicle:

water

Details on 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 La-boratory 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.

Details on mating procedure:

- M/F ratio per cage:1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: Vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted " gestation day (GD) 0" and the following day "gestation day (GD) 1"

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)

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

12 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

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

Maternal examinations:

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

Fetal examinations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups,

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead]

OTHER: On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood were sampled for determination of thyroid hormone con-centrations. All pups were examined externally and eviscerated; their organs were assessed macroscopically.

Statistics:

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

Indices:

live birth index; postimplatation loss; viability index; lactation index, sex ratio; anogenital index

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.

Dermal irritation (if dermal study):

not examined

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 (No. 127 - 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 concurrent 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) 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.

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.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no indications for test substance-induced intrauterine embryo-/fetolethality since the post-implantation loss did not show any significant differences between the groups (13.0 / 11.0 / 6.0 and 13.3 mean% in test groups 00 - 03, respectively), and the mean number of F1 pups delivered per dam remained unaffected (11.4 / 11.3 / 12.0 and 11.4 pups/dam in test groups 00 - 03, respectively).

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (13.1 / 12.8 / 12.8 and 13.1 implants/dam in test groups 00 - 03, respectively).

Dead fetuses:

no effects observed

Description (incidence and severity):

The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 97.4% / 100% / 97.5% and 98.2% in test groups 00 - 03, respectively. Moreo-ver, the number of stillborn pups was not significantly different between the test groups.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

The mean duration of gestation values varied between 22.1 / 22.1 / 22.1 and 22.3 in test groups 00 - 03, respectively.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: high dose

Fetal body weight changes:

no effects observed

Description (incidence and severity):

- F1 pups
The mean body weights and body weight change of all male and female pups in all test substance-treated groups were comparable to the concurrent control values throughout the entire study.
One male and one female runt was seen in the control, one male and three female runts were seen in test group 02 and one female runt was seen in test group 03.

- F1 rearing animals
Mean body weights and body weight change of all male and all female animals in all test substance-treated groups were comparable to the concurrent control values during their entire study.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The viability index indicating pup survival during lactation (PND 0 - 4) varied between 100%/ 98.6% / 99.3% and 100% in test groups 00 - 03, respectively.The pups surviving index indicating pup survival during lactation (PND 4 - 21) varied be-tween 73.5%/ 75% / 75% and 73.8% in test groups 00 - 03, respectively. These figures factor in the scheduled sacrificed pups on PND 13 for blood sampling for hormone measurement. Thus, the test substance did not influence pup survival in any of the treated groups (12, 40 and 120 mg/kg bw/d).

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution and sex ratios of live F1 pups on the day of birth and PND 21 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The anogenital distance and anogenital index of all test substance treated male and female pups were comparable to the concurrent control values.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

The pups surviving index indicating pup survival during lactation (PND 4 - 21) varied be-tween 73.5%/ 75% / 75% and 73.8% in test groups 00 - 03, respectively. These figures factor in the scheduled sacrificed pups on PND 13 for blood sampling for hormone measurement.

External malformations:

no effects observed

Description (incidence and severity):

A few pups showed spontaneous findings at gross necropsy, such as diaphragmatic hernia and dilated renal pelvis. These findings occurred without any relation to dosing. Thus, all these findings were considered not to be associated to the test substance.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: high dose

Abnormalities:

no effects observed

Developmental effects observed:

no

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.

Conclusions:

Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the oral administration 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.
The NOAEL for fertility and reproductive performance was 120 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was 120 mg/kg bw/d.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

120 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

155.6 mg/m³

Study duration:

subacute

Species:

rabbit

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Data are available for 4-hydroxybutyl acrylate. In addition data from the structurally analogous substances 2-hydroxyethyl acrylate (CAS No. 818-61-1), hydroxypropyl acrylate (CAS No. 25584-83-2) and methyl acrylate (CAS No. 96-33-3) developmental toxicity studies in rats and rabbits are available.

Data for 4 -HBA CAS No. 2478 -10 -6 (combined OECD 408/422 by oral gavage):

4-HBA 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). 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 dose selection was based on the results of 14 d range-finding studies (0; 50; 150; 200 and 600 mg/kg bw/d), the animals of the 600 mg/kg bw/d were sacrificed moribund / died prematurely, therefore, organ weights were not recorded. Significantly increased absolute and relative liver weights were described at 150 mg/kg bw/d and 200 mg/kg bw/d, dose-dependent adverse local effects to forestomach and stomach occurred at 150 mg/kg bw/d and higher.

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.

After 10 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 were detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females.

A detailed clinical observation (DCO) was performed in all F0 animals before the start of the administration period and, as a rule, thereafter at weekly intervals for the F0 animals and F1 rearing animals. Water consumption of the F0 parents during premating and F1 rearing animals was determined once a week (over a period of 3 days). However, during gestation and lactation water consumption of the F0 females were determined on gestation days (GD) 0-1, 7-8, 13-14, and 19-20 and on postnatal days (PND) 1-2, 3-4, 6-7, 9-10, 12-13, 16-17 and 20-21. Food consumption of the F0 parents during premating and F1 rearing animals was determined once weekly. In dams food consumption was determined for GD 0-7, 7-14, 14-20 and PND 1-4, 4-7, 7-10, 10-13, 13-17 and 17-21. In general, body weights of F0 parents and F1 rearing animals were determined once weekly. However, during gestation and lactation F0 females were weighed on GD 0, 7, 14 and 20 and on PND 0, 1, 4, 7, 10, 13, 17 and 21. Estrous cycle data were evaluated for F0 generation females over a three weeks period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1, 4, 7, 13 and 21. Their viability was recorded. At necropsy, all pups were sacrificed with CO₂under isoflurane anesthesia (except the selected pups for blood sampling) and examined macroscopically for external and visceral findings.

Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1.All surviving male pups were examined for the presence or absence of nipple/areola anlagen on PND 12. If nipple/areola anlagen were recorded, all surviving male pups were carefully re-examined one day prior to necropsy (PND 20).

Blood samples were taken from all surplus pups at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia for hormone measurement. Clinico-chemical and hematological examinations were performed in 5 F0 animals per sex and group towards the end of the administration period. Blood samples from all dams at PND 14 and all F0 males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement. Urine samples for clinical pathological investigations were withdrawn from all F0 parents. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group. Various sperm parameters (motility, sperm head count, morphology) were assessed in the male F0 parental animals at scheduled sacrifice or after appropriate staining. 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.

 

The following test substance-related relevant effects/findings were noted in the F0 parental animals at 120 mg/kg bw/d:

• Salivation after gavage dosing in all male animals during the entire study and in all female animals during premating, gestation and lactation period, and in 2 females during mating period
• Reduced prothrombin time in F0 males
• Decreased LDL-cholesterol in F0 males
• Increased inorganic phosphate values in males
• signs of erosion/ulcer in the forestomach one male and two females
• focal hyperplasia of the margo plicatus of four males and four females
• vacuolation of the hepatocytes in the periportal area of the liver of males

 

No test substance-related, adverse findings (also including macroscopically for external and visceral findings) were observed at F1 pups and rearing animals at 120 mg/kg bw/d. No test substance-related, adverse findings were observed in the F0 animals and F1 pups and rearing animals at 15 and 40 mg/kg bw/d.

 

In conclusion, under the conditions of the present OECD 408/422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the oral administration of 4HBA 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. The NOAEL for fertility and reproductive performance was 120 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was 120 mg/kg bw/d (BASF, 2022)

Data from the structural analogue 2-hydroxyethyl acrylate (CAS No. 818-61-1)

In a prenatal developmental toxicity study, Hydroxyethyl acrylate was administered to pregnant New Zealand White rabbits daily by stomach tube from implantation to one day prior to the expected day of parturition (GD 6-28).

Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle.

Clinical examinations revealed no toxicologically relevant difference between the animals receiving 6 or 20 mg/kg bw/d Hydroxyethyl acrylate and the controls.

At the high-dose level of 60 mg/kg bw/d slightly, but consistently lower food consumption as well as lower body weight gain during the first two treatment weeks may represent beginning signs of maternal toxicity.

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 60 mg/kg bw/d.

Regarding pathology, all 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.

There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and postimplantation losses, the numbers of resorptions and viable fetuses. Similarly, no influence of the test substance on uterine weight, placental weight, fetal weight and sex distribution of the fetuses was noted at any dose. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

Fetal examinations revealed no toxicologically relevant adverse effects of the test substance on embryofetal development.

In a study where the developmental toxicity of seven acrylates was investigated (Saillenfait, 1999), groups of 25 pregnant rats were exposed to 0, 1, 5 or 10 ppm 2-hydroxyethyl acrylate vapour (corresponding to approx. 0.0048, 0.0241, and 0.0482 mg/L) for 6 hrs/day from days 6 through 20 of gestation. Maternal toxicity was demonstrated at 10 ppm as a statistically significant decrease in maternal body weight gain over the entire exposure period, which was also statistically different from controls on days 6-13. A statistically significant decrease in food consumption as compared to controls was also observed for the 10 ppm group on days 6-21. Uteri were removed and weighed, and the number of implantation sites, resorptions, and dead and live fetuses were recorded. Uteri which had no visible implantation sites were stained with ammonium sulfide to detect very early resorptions. Live fetuses were weighed, sexed, and examined for external anomalies including those of the oral cavity. Half of the live fetuses from each litter were preserved in Bouin’s solution and examined for internal soft tissue changes. The other half were fixed in ethanol, eviscerated, and then processed for skeletal staining with alizarin red S for subsequent skeletal examination. There were no treatment-related increases in the number of implants and the number of live fetuses, in the incidence of non-live implants and resorptions, or fetal body weights across groups. The only malformation observed was a unilateral microphthalmia at 1 ppm. There were no significant changes in the incidence of external, visceral, or skeletal variations. The NOAEC for maternal toxicity was 5 ppm (= approx. 0.0241 mg/L); the NOAEC for developmental effects and fetotoxicity was 10 ppm (= approx. 0.0482 mg/L).

Data from the structural analogue hydroxypropyl acrylate (CAS No. 25584-83-2)

In the same study groups of 25 pregnant rats were exposed to 0, 1, 5 or 10 ppm hydroxypropyl acrylate vapour (corresponding to approx. 0.0054, 0.027, and 0.054 mg/L) for 6 hrs/day from days 6 through 20 of gestation (Saillenfait et al., 1999). Maternal body weights were lower than control for the 10 ppm group and weight gain was reduced for the 5 and 10 ppm groups. There was no significant difference in the numbers of implantation sites and live fetuses, in the incidence of non-live implants and resorptions, or in the fetal sex ratio, or fetal body weight between control and treated animals. No treatment related increase in embryo/foetal lethality or fetal malformations was observed at any dose level. The incidence of external, visceral, and skeletal variations was similar to controls. The NOAECs were 1 ppm (0.0054 mg/L) for maternal toxicity and 10 ppm (0.054 mg/L) for embryo-foetal toxicity and teratogenicity. In addition, developmental studies in rabbits are available for the structural analogue methyl acrylate (CAS No. 96-33-3).

Data from the structural analogue methyl acrylate (CAS No. 96-33-3)

A prenatal developmental toxicity study in rabbits as second species was conducted according to OECD TG 414 for the Acrylate Task Force (BASF, 2009). 25 inseminated female Himalayan rabbits per group were whole-body exposed for 6 hrs/day, 5 days/week over a time period of 23 consecutive days (gestation days (GD) 6–28) to methyl acrylate vapours at target concentrations of 0, 5, 15, and 45 ppm. Analytical concentrations of 4.9, 15.7, 44.2 ppm (corresponding to approx. 0.0174, 0.0553, 0.1556 mg/L) were measured. On gestation day 29 the does were sacrificed and submitted to gross and histopathological examination (nasal cavities, larynx, trachea, lungs, mediastinal lymph nodes, all gross lesions). Examinations of ovaries and uterine content of the does included: determination of the weight of the unopened uterus, of the number of corpora lutea, of the number and distribution of implantation sites, and calculations of conception rate and pre- and post-implantation losses. Fetal examinations were performed on all fetuses per litter (external, soft tissue, skeletal) except head examinations that were done on half of the fetuses per litter. There were no test substance-related effects on the does concerning food consumption, gross/net body weight, gestational parameters, uterine, placental and lung weights, as well as necropsy observations up to and including a dose of 45 ppm. The test substance caused a severe degeneration and atrophy of the olfactory epithelium at at least one focal area in the nasal cavity (distal levels III and/or IV) at the high-dose level (45 ppm). Though being local effects, such massive findings in the respiratory tract are likely to cause a considerable amount of distress in the affected maternal animals. Since distress is supposed to influence maternal homeostasis, this is considered to be a significant adverse effect on the maternal organism. The NOAEC for maternal toxicity was 15 ppm (0.0553 mg/L). Fetal examinations revealed no influence of the test compound on sex distribution of the fetuses and fetal body weights. Methyl Acrylate (MA) had no adverse effect on prenatal development of offspring at any of the dose levels tested (5, 15 and 45 ppm). Thus, the NOAEC for developmental effects (fetotoxicity) and the NOAEC for developmental effects (teratogenicity) was the highest concentration tested of 45 ppm (0.1556 mg/L).

Data from the structural analogue n-butyl acrylate (CAS No. 141-32-2):

An oral developmental toxicity study in rabbits according to OECD414 and GLP was performed with n-butyl acrylate (Acrylate REACH TF, 2017).

25 inseminated New Zealand White rabbits were exposed orally (gavage) to 50, 150 and 400 mg/kg bw/day during gestation days 7 through 28. All females in the control, 50, 150, and 400 mg/kg/day groups survived to the scheduled necropsy. No test substance-related clinical observations were noted at the daily examinations or approximately 1 hour following dose administration at any dosage level.

A test substance-related absence of a mean body weight gain (0 g) was noted in the 400 mg/kg/day group on the first day of dose administration (gestation day 7-8) and resulted in a 78.9% lower mean body weight gain in this group compared to the control group during gestation days 7-10 and a lower mean body weight gain when the entire treatment period (gestation days 7-29) was evaluated. However, the aforementioned differences were not statistically significant and were not of sufficient magnitude to affect mean body weights at this dosage level, and therefore were considered test substance-related but nonadverse. In addition, lower mean food consumption was noted in the 400 mg/kg/day group during gestation days 7-10 and resulted in lower mean food consumption in this group compared to the control group when the entire treatment period (gestation days 7-29) was evaluated; however, these differences were not statistically significant and were not of sufficient magnitude to affect mean body weights at this dosage level, and therefore were considered test substance-related but nonadverse. Mean maternal body weights, body weight gains, and food consumption in the 50 and 150 mg/kg/day groups and mean body weights,net body weights, net body weight gains, and gravid uterine weights in the 50, 150, and 400 mg/kg/day groups were unaffected by test substance administration. There were no substance-related macroscopic findings noted at the scheduled necropsy on gestation day 29 in the 50, 150, and 400 mg/kg/day groups. Intrauterine growth and survival in the 50, 150, and 400 mg/kg/day groups were unaffected by test substance administration. In addition, no test substance-related external, visceral, and skeletal malformations or developmental variations were noted at any dosage level.

Nonadverse lower mean body weight gains and corresponding lower mean food consumption were noted in the 400 mg/kg/day group. No evidence of developmental toxicity was noted at 50, 150, and 400 mg/kg/day. Based on these results, a dosage level of 400 mg/kg/day, the highest dosage level tested, was considered to be the no‑observed‑adverse‑effect level (NOAEL).

Sprague Dawley rats were exposed to butyl acrylate vapour concentrations of 25, 135 and 250 ppm (corresponding to approx. 0.13; 0.71; 1.31 mg/L) for 6 hours per day on days 6 to 15 of gestations (BASF AG, 1979). Inhalation of 135 and 250 ppm caused a significant reduction in maternal body weight gain, as well as irritation to the nose and eyes due to the known irritative nature of this test substance to mucous membranes. Following the exposure until termination the animals recovered and the weight gain was comparable to the controls. The two highest concentrations caused a dose-dependent increase in post-implantation loss (i.e. increase in resorptions). 25 ppm did not lead to any signs of maternal toxicity and had no effect on the implants. No substance-related morphogical alterations, especially no malformations were observed in the fetuses at any concentration. The NOAEC for maternal and developmental toxicity was 25 ppm (0.13 mg/L), and the NOAEC for teratogenicity was 250 ppm (1.31 mg/L), the highest concentration examined. It is noteworthy to mention that no histopathological examination of the respiratory tract of the dams was performed, as it was not required by the guideline. However, with regards to the observed respiratory irritation in the subchronic inhalation study at comparable concentrations, the occurrence of clear effects on the respiratory tract as evidence for maternal toxicity can be considered as very likely, especially as irritation was already noted clinically.

Subsequently, a comparative prenatal developmental toxicity study equivalent to OECD 414 on seven acrylates was performed in the same strain of Sprague-Dawley rats with a prolonged exposure period (Saillenfait, 1999). Groups of 25 pregnant rats were exposed to 0, 100, 200 and 300 ppm butyl acrylate (corresponding to approx.0.52, 1.05, and 1.57 mg/L) for 6 hrs/day from day 6 through day 20 of gestation. The body weight gain was concentration-dependent reduced in all dose groups. In contrast to the above study, no treatment-related effects occurred for the numbers of implantation sites, live fetuses, non-live implants or resorptions but fetal body weight was significantly reduced at 200 and 300 ppm. There were no treatment-related effects on the incidence of external and visceral variations. The incidence of individual skeletal variations was similar in the control and treated groups. A NOAEC for maternal toxicity could not be derived with regards to the impaired body weight gain, the LOAEC for was 100 ppm (0.52 mg/L). The NOAEC for developmental effects was 100 ppm (0.52 mg/L), and the NOAEC for teratogenicity was the highest tested concentration of 300 ppm (1.57 mg/L).

Conclusion

In developmental studies conducted with 4HBA itself and the structural analogues 2-hydroxyethyl acrylate and hydroxypropyl acrylate in rats by the inhalation route, no signs of developmental toxicity (fetotoxicity, embryotoxicity, teratogenicity) were observed. This is supported by a developmental study in rabbits as non-rodent species with the structural analogue methyl acrylate. Based on the structural similarities between the substances, it can be safely assumed that 4-hydroxybutyl acrylate does not cause developmental toxicity.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No 1272/2008. Based on the data, classification for toxicity to reproduction is not warranted under Regulation (EC) No 1272/2008.