Registration Dossier

Administrative data

Description of key information

In a GLP-compliant oral 90-day repeated dose toxicity study according to OECD Guideline 408 the NOAEL (no observed adverse effect level) for systemic toxicity of Hydroxypropylacrylate was 100 mg/kg bw/d (highest tested dose) for male and female Wistar rats.

In addition, there are data on the structural analogue 2-hydroxyethyl acrylate available:

Following chronic exposure by the inhalation route (5 d/w, 6 h/d), the NOAEC in Sprague-Dawley rats was 0.0024 mg/L (nominal) based on an increased incidence, increased severity, and earlier onset of the lesions associated with chronic murine pneumonia. Gross and histopathological examination of tissues showed no indication of significant chronic toxicity.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 May - 29 Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No. of test material: A022I34007
- Purity: 97.84 wt%
- Water content: 0.06 wt%
- Manufacturing date: 2018-03-04

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (room temperature)
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Solubility and stability of the test substance in the solvent/vehicle: Solubility analyses performed previously demonstrated that the formulation was soluble in the vehicle at a concentration of 12.0 mg/mL. Stability analyses performed previously demonstrated that the test substance is stable in the vehicle following at least 49 hours of room temperature storage, and following at least 8 days of refrigerated storage in formulations at concentrations bracketing those used in the present study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dilution in vehicle
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species and strain for toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Females (if applicable) nulliparous and non-pregnant: no information available
- Age at study initiation: approx. 7 weeks
- Weight at study initiation: 139 - 270 g
- Housing: in groups of 3 animals of the same sex until randomization; in groups of 2 animals of the same sex and dosing group following randomization
- Diet: ad libitum; PMI Nutrition International, LLC Certified Rodent LabDiet® 5CR4 meal
- Water: ad libitum; municipal tap water after treatment by reverse osmosis
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water was performed. It was considered that there were no known contaminants in the water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): >= 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 17 May 2018 To: 29 Aug 2018
Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected since oral ingestion is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.
Vehicle:
water
Remarks:
deionized
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test substance dosing formulations were prepared based on Sponsor instructions at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily during Week 1 and approximately weekly thereafter. An adequate amount of each formulation was dispensed into daily aliquots, which were stored in a refrigerator set to maintain a target of 5°C, until use. The dosing formulations were stirred continuously during dosing.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis:
Day -1: Group 1
Day 1: Groups 2 - 4
Day 4: Groups 2 - 4
Day 28: All groups
Day 56: All groups
Day 84: All groups
Analyses were performed by a gas chromatography method with flame ionization detection using a validated analytical procedure.

The analyzed dosing formulations contained 90.2% to 110% of the test substance which was within the protocol-specified range of target concentrations for solutions (90% to 110%) with the following exceptions. The mean analyzed concentrations of the 30 May 2018 Group 2, Group 3, and Group 4 formulations were 118%, 87.3%, and 84.9% of the target concentrations, respectively. The back-up samples were processed and analyzed on 31 May 2018. The mean
analyzed concentration of the Group 2 formulation met the previously stated protocol-specified acceptance criteria for concentration. The results of the analyzed Group 3 and Group 4 back-up samples confirmed the initial results. This is not expected to have an impact on the study since the formulations were being prepared daily at the time, and the samples collected on Day 4 were within the acceptable range. In addition, the mean analyzed concentrations of the 26 Jun 2018 Group 2 and Group 3 formulations were 89.1% and 84.0% of the target concentration, respectively. Although formulations were being prepared approximately weekly at this sampling interval, this is not expected to impact the study since the analyzed values for Groups 2 and 3 were within 20% of the targeted concentration and Group 4 samples met acceptance criteria.
No test substance was detected in the analyzed vehicle administered to the control group (Group 1).
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose selection was based on the results of an OECD TG 422 in Wistar rats, in which strong irritation including erosion and ulcer in the forestomach, as well as inflammatory changes in the duodenum were detected in the parental animals of the high dose (150 mg/kg). The effects in the forestomach and duodenum were still observed in the mid dose (50 mg/kg), but less pronounced. It was anticipated that the high-dosage level would show substance-specific effects but not produce an incidence of fatalities that would prevent a meaningful evaluation. The lower dosage levels were selected at intervals that were predicted to be narrow enough to reveal any dose-related trends. Though priority was given to detecting a dose-related trend, it was expected that the low-dosage level would be a no-observed-adverse-effect level (NOAEL).

- Fasting period before blood sampling for clinical biochemistry: overnight
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes (for general health/mortality and moribundity)
- Time schedule: twice daily, once in the morning and once in the afternoon
- Cage side observations were performed daily, beginning on Day 1 and lasting throughout the dosing period. During the dosing period, these observations were performed 1 to 3 hours postdose.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: within 4 days of receipt, on the day of randomization, on Day 1 (prior to dosing), weekly (± 2 days) during the study period, and on the day of the scheduled necropsy
- Examined parameters: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size (further parameters as required)

BODY WEIGHT: Yes
- Time schedule for examinations: within 4 days of receipt, on the day of randomization, on Day 1 (prior to dosing), weekly (± 2 days) during the study period, on the day prior to the first day of scheduled necropsy, and on the day of the scheduled necropsy. A fasted weight was recorded on the day of the scheduled necropsy.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: weekly (± 2 days) starting on Day 1 and continuing weekly throughout the study

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during the acclimation period (Day -9) and near the end of the dosing period (Day 87)
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13 (Day 91 or 92)
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters checked; Total leukocyte count (WBC); Erythrocyte count (RBC); Hemoglobin (HGB); Hematocrit (HCT); Mean corpuscular volume (MCV); Mean corpuscular hemoglobin (MCH); Mean corpuscular hemoglobin concentration (MCHC); Platelet count (Platelet); Reticulocyte count; Percent (RETIC); Absolute (RETIC Absolute); Differential leukocyte count - Percent and absolute; -Neutrophil (NEU); -Lymphocyte (LYMPH); -Monocyte (MONO); -Eosinophil (EOS); -Basophil (BASO); -Large unstained cell (LUC); Red cell distribution width (RDW); Platelet estimate; Red cell morphology (RBC Morphology)
Activated partial thromboplastin time; Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13 (Day 91 or 92)
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters checked: Alanine aminotransferase (ALT); Albumin; Albumin/globulin (A/G) ratio (calculated); Alkaline phosphatase (ALP); Aspartate aminotransferase (AST); Calcium; Chloride; Creatinine; Gamma glutamyltransferase (GGT); Globulin (calculated); Glucose; Phosphorus; Potassium; Sodium; Sorbitol dehydrogenase (SDH); Total bilirubin; Total cholesterol; Total protein; Triglycerides; Urea nitrogen; Appearance;

URINALYSIS: Yes
- Time schedule for collection of urine: Week 13 (Day 91 or 92)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (during stay in the metabolism cages)
- Parameters checked: Bilirubin; Color and clarity; Glucose; Ketones; Occult blood; pH; Protein; Specific gravity; Total volume

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment (see "OTHER")

OTHER:
Functional observational battery (FOB): for all animals/sex/group during Week 13 prior to exposure
- The functional observational battery was carried out in 10 animals/sex/group during Study Week 12.
- Examined parameters:
• Home cage observations: Biting, Convulsions/tremors, Feces consistency, Palpebral (eyelid) closure, Posture
• Handling observations: Ease of removal from cage, Lacrimation/chromodacryorrhea, Piloerection, Palpebral closure, Eye prominence, Red/crusty deposits, Ease of handling animal in hand, Salivation, Fur appearance, Respiratory rate/character,
Mucous membranes/eye/skin color, Muscle tone
• Open field observations (evaluated over a 2-minute observation period): Mobility, Rearing, Convulsions/tremors, Grooming, Bizarre/stereotypic behavior, Time to first step (seconds), Gait, Arousal, Urination/defecation, Gait score, Backing
• Sensory observations: Approach response, Startle response, Pupil response, Forelimb extension, Air righting reflex, Touch response, Tail pinch response, Eyeblink response, Hindlimb extension, Olfactory orientation
• Neuromuscular observations: Grip strength-hind and forelimb, Hind limb extensor strength, Hind limb foot splay, Rotarod performance
• Physiological observations: Body temperature, Body weight, Catalepsy

Motor activity assessment:
- assessed for all animals/sex/group during Week 13
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- examination of the external surface, all orifices and the cranial, thoracic, abdominal and pelvic cavities, including viscera.

HISTOPATHOLOGY: Yes
- Microscopic examination of hematoxylin-eosin stained paraffin sections was performed from animals in the control and high-dose groups at the scheduled necropsy.

Organs weighed: Adrenal glands; Brain; Epididymides (total and caudal); Heart; Kidneys; Liver; Ovaries; Pituitary; Prostate with seminal vesicles; Spleen; Testes; Thymus; Thyroid with parathyroid; Uterus

Tissue collection and preservation: Adrenal glands; Aorta; Bone with marrow; Sternum; Femur; Bone marrow smear (from femur); Brain; Cervix; Epididymides; Eyes with optic nerves; Gastrointestinal tract; Esophagus; Stomach; Duodenum; Jejunum; Ileum; Cecum; Colon; Rectum; Harderian glands; Heart; Kidneys; Larynx; Liver (sections of 2 lobes); Lungs (including bronchi, fixed by inflation with fixative); Lymph nodes; Axillary; Mesenteric; Mandibular; Ovariesf with oviductsd; Pancreas; Peripheral nerve (sciatic); Peyer's patches; Pharynx; Pituitary; Prostate; Salivary glands (mandibular); Seminal vesicles; Skeletal muscle (rectus femoris); Skin with mammary glande; Spinal cord (cervical, thoracic, lumbar); Spleen; Testes; Thymus;Thyroid (with parathyroids; Tongue; Trachea; Uterus; Urinary bladder; Vagina; Gross lesions (when possible)
Other examinations:
Coagulation parameters: at the time of euthanasia from animals euthanized via carbon dioxide inhalation

Spermatogenic evaluations: The following quantitative assessment of the process of spermatogenesis was performed on all surviving males at the scheduled necropsy.
- Motility/viability assessment
- Morphology assessment
- Enumeration of epididymal and testicular sperm and sperm production rate (SPR)

Stage-dependent qualitative light microscopic evaluation of spermatogenesis was conducted on sections of testicular tissues with special attention given to the normal progression of stages of the spermatogenesis cycle, cell associations, and cell proportions expected to be present during spermatogenesis.

Thyroid Hormone Assessments:
- Blood samples were collected from all study animals for analysis of T3, T4, and TSH levels on the day of the terminal necropsy.

On the day of scheduled necropsy, all females received a vaginal lavage to determine the stage of estrus.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and are reported at the 1% and 5% levels.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Datasets were compared using an overall one-way ANOVA F-test.12 If the overall F-test was found to be significant, then the above pairwise comparisons were conducted using Dunnett’s test.
Further details are given in table 1.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- body weights/body weight gains were lower than expected from Days 85–90 in both sexes and all groups, which could be due to a brief period of fasting for the functional observational battery evaluations.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- test substance-related higher mean food consumption in the 100 mg/kg/day group males and females throughout the dosing period (Day 1–90) and in the 30 mg/kg/day group males from Day 29 through Day 90 (frequently statistically significant)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- statistically significantly higher mean cholesterol and potassium, and lower mean chloride levels in the 100 mg/kg/day group males on Week 13
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- Home cage observations were unaffected by test substance administration. The only statistical significant difference from the control group was a higher number of females in the 10 m/kg/day group without fecal pellets. Based on a lack of dose response, this finding was not considered to be test substance-related.

- Handling observations, open field observations, sensory observations, neuromuscular observations and physiological observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the Week 13 evaluation.
- Motor activity patterns (total and ambulatory activity counts) were unaffected by test substance administration. Values obtained from the 6 epochs evaluated (0-10 minutes, 11-20 minutes, 21-30 minutes, 31-40 minutes, 41-50 minutes, and 51-60 minutes) and the overall 60-minute test session were comparable to the concurrent control values. No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups when the animals were evaluated on Week 13.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- statistically significantly higher mean liver weights (absolute and relative to body and brain weights) and higher mean absolute and relative thyroid gland weights (not statistically significant) in the 100 mg/kg/day group males. There were no corresponding microscopic alterations in the liver or thyroid gland, thus, the weight changes were considered test substance-related and non-adverse.
- higher mean organ to body weight ratio values for the liver and thyroid/parathyroid in the 100 mg/kg/day group males, compatible with a test substance-related effect.
- higher mean kidney weights in the 100 mg/kg/day group males and lower mean thymus weights in the >10 mg/kg/day group females, but relationship of these to the test substance was considered uncertain (absence of microscopic changes)
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No test substance-related changes in mean T3, T4, or TSH levels were detected on this study.
No test substance-related changes in sperm morphology or differential counts were detected on this study
There was no test substance-related effect on ovarian follicle counts in the 100 mg/kg/day group compared to the control group
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to and including the highest dose group
Critical effects observed:
no

For summary tables of body weight, body weight changes, hematology and coagulation values, serum chemistry values, other chemistry values, sperm motility and concentrations, sperm morphology differential counts, gross pathology findings, organ weights absolute and relative, and histopathology findings see "Attached background material".

 

Conclusions:
Based on the results of this study, oral administration of the test substance to Wistar Han rats at dosage levels of 10, 30, and 100 mg/kg/day for a minimum of 90 days was well tolerated at all dosages. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 100 mg/kg/day.
Executive summary:

The test substance was administered daily by oral gavage to 10 male and 10 female Wistar Han rats at doses of 0 mg/kg bw/day (test group 1), 10 mg/kg bw/day (test group 2), 30 mg/kg bw/day (test group 3) and 100 mg/kg bw/day (test group 4) for at least 90 consecutive days.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, functional observational battery, motor activity, ophthalmology, thyroid hormone assessment, clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis), spermatogenesis evaluation, gross necropsy findings, organ weights, and histopathologic examinations.

All animals survived to the scheduled necropsy. There were no test substance-related clinical observations or effects on body weight, hematology, coagulation, or urinalysis. There were no test substance-related ophthalmic, macroscopic, microscopic findings, or effects on ovarian follicle counts, thyriod hormones (T3, T4 or TSH levels), sperm morphology or differential counts.

Test substance-related higher food consumption was noted in the 100 mg/kg/day group males and females (Days 1–90), and in the 30 mg/kg/day group males (Days 29–90), compared to the control group.

Test substance-related higher cholesterol and potassium, and lower mean chloride levels were noted in the 100 mg/kg/day group males on Week 13, compared to the control group.

Test substance-related higher mean organ weights were noted in the liver and thyroid/parathyroid gland of the100 mg/kg/day group males. Higher mean kidney weights were present in the 100 mg/kg/day group males, and lower mean thymus weights were seen in the >10 mg/kg/day group females, but relationship of these to the test substance was considered uncertain.

Based on the results of this study, oral administration of the test substance to Wistar Han rats at dosage levels of 10, 30, and 100 mg/kg/day for a minimum of 90 days was well tolerated at all dosages. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 100 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
guideline study

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: inhalation
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 May 1974 - 07 May 1976 (experimental)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well documented study report, acceptable for assessment with restrictions. In the study report, test parameters didn’t totally comply with the specific testing guideline, but were sufficient to accept data and study was conducted prior to the advent of GLP regulations (1982).
Principles of method if other than guideline:
Groups of 99-100 male and female rats were erposed to atmospheres containing 0 ppm (controls), 5.0 ppm (24 mg/m3) or 0.5 ppm (2.4 mg/m3) 2-hydroxyethyl acrylate (HEA) for 6 hours/day, 5 days/week over an 18 month period, and subsequently held for a post-exposure period of 5 months (males) and 6 months (females). The study included 12-month interim sacrifices for pathologic and cytogenetic examinations.
GLP compliance:
no
Remarks:
(Study was conducted prior to the advent of GLP regulations)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Spartan substrain
- Source: Spartan Research Animals, Michigan
- Housing: 3-4 animals/cage
- Diet: ad libitum
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): ambient
- Humidity (%): ambient
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 3.7 cubic meters stainless steel chambers under dynamic airflov- conditions
- System of generating vapours: The exposure atmosphere in each chamber was generated by metering liquid HEA at a calculated rate into the top of
a 15 inch glass column ( 2" diameter) that was heated to a temperature (ca. 80°C) hot enough to vaporize the HEA. Dry compressed air was introduced at the bottom of the glass column to sweep the vapours into the chamber where they were diluted with room air at a rate calculated to provide the desired HEA Concentration.


TEST ATMOSPHERE
- The nominal concentration of HEA in the chamber was calculated from the ratio of the amount of liquid HEA used to the rate of total chamber airflow.
- Brief description of analytical method used:
The concentration of HEA in each chamber was determined three or more times daily.
Analytical concentrations for the first 6 1/2 months of exposure were obtained by drawing 10 liters of air from the chamber at a rate of 1 liter/min through a charcoal tube. The HEA absorbed on the charcoal was extracted into 2 mL of carbon disulfide. The quantity of HEA in a 2 µL sample of carbon disulfide extract was analysed by gas chromatography (detector: FID).
For the last 11 1/2 months of the study HEA samples were collected by bubbling chamber air through water instead of charcoal. Improved reproducibility of sample analysis and convenience were the primary reasons for using water instead of charcoal. Fifty liters of air from the chamber were drawn through 20 mL of distilled water in a fritted glass bubbler at a rate of 1 liter/min. The quantity of HEA in a 2 µL sample of the trapping solution was analyzed by gas chromatography using the same conditions as before.
- Samples taken from breathing zone: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sixty per cent of the total exposure days for the low exposure level and 73 % of the total exposure days of the high exposure level were within 50 % of the an analytical concentrations. The high nominal concentration values and variability in analytical concentrations reflect the fact that HEA, having a relatively low vapour pressure, was difficult to vaporize. Much of the HEA dispensed into the vaporization apparatus was not vaporized, but that not vaporized was not subtracted from the amount dispensed in calculation of the nominal concentration. Although the analytical concentrations were not identical to the target concentrations, especially for the higher exposure level, the target concentrations of 0.5 and 5.0 ppm were referred to throughout the study report.
Duration of treatment / exposure:
18 months
Frequency of treatment:
6 hours per day, 5 days per week
Dose / conc.:
0.024 mg/L air (nominal)
Remarks:
corresponding to 5 ppm)
Dose / conc.:
0.002 mg/L air (nominal)
Remarks:
corresponding to 0.5)
No. of animals per sex per dose:
99 - 100
Control animals:
yes, sham-exposed
Details on study design:
- Post-exposure recovery period: Males: 5 months; females: 6 months
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: no details given


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: no details given


BODY WEIGHT: Yes
- Time schedule for examinations: no details given


HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to the scheduled 12-month interim sacrifice, and again after 5 months of the post-exposure observation period
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 rats/sex/exposure group
- Parameters were examined: packed cell volume (PVC), red blood cell count (RBC), hemoglobin concentration (Hgb), total white blood cell count (WBC) and differential white blood cell count.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of serum: prior to the scheduled 12-month interim sacrifice, and again after 5 months of the post-exposure observation period
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 5 and 9 or 10 rats/sex/exposure group, respectively
- Parameters were examined: blood urea nitrogen (DUN) concentration, serum alkaline phosphatase (AP) activity, and serum glutamic pyruvic transaminase (SGPT) activity


URINALYSIS: Yes
- Time schedule for collection of blood: prior to the scheduled 12-month interim sacrifice, and again after 5 months of the post-exposure observation period
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 5 and 10 rats/sex/exposure group, respectively
- Parameters were examined: Urinary specific gravity, pH, and the presence or absence of glucose, protein, ketones, bilirubin and occult blood were evaluated at both time intervals. Urinary urobilinogen was evaluated at the preterminal sampling interval only.



Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

An interim necropsy of 5 rats/sex/exposure group was conducted after 12 months exposure. Terminal necropsy was conducted after completion of
23 months (18 months exposure + 5 months post-exposure) for male rats, and after completion of 24 months (18 months exposure + 6 months post-exposure) for female rats.

The eyes of 5 rats/sex/exposure group were placed in Zenker's fixative. The trachea and lungs were removed as a unit and distended with formalin
fixative. The weights of the brain, heart, liver, kidneys and testes (males) were recorded for 5 rats/sex/group sacrificed after 12 months and also for 9-19 rats/sex/exposure group sacrificed at termination.

Gross examination:
Representative portions of the major organs and tissues, including brain, heart, liver, kidneys, testes, lungs, thoracic and/or mesenteric lymph nodes, salivary glands, pancreas, adrenals, spleen, thymus, aorta, skeletal muscle, small intestine, large intestine, thyroid gland, parathyroid glands, eyes (those not fixed in Zenker's fixative), esophagus, trachea, spinal cord, peripheral nerve, pituitary gland, epididymides, urinary bladder, accessory sex glands, adipose tissue, ovaries, uterus, nasal turbinates, and any gross lesion suggestive of an unexpected pathologic process or with a tumour formation was preserved in buffered 10 % formalin fixative. The eyes were examined with a glass slide technique and the data were entered with the gross data.

Microscopic examination:
- Control and High (5.0 ppm) Exposure Group: All available tissues from all rats were examined.
- Low (0.5 ppm) Exposure Group: In the absence of any discernible exposure-related lesions in the tissues from rats exposed to 5.0 ppm, the 12-month interim examination of rats of the 0.5 ppm exposure group was limited to grossly visible lesions suggestive of tumour formation. From the terminal necropsy, all available lungs, livers, kidneys, lymph nodes, tracheas, plus grossly visible lesions suggestive of an unexpected pathologic process
or tumour formation were examined from all rats.
Statistics:
Significance of differences between control and test values for hematology, clinical chemistry, body weights, organ weights, and organ/body weight ratio data was statistically determined by an analysis of variance and Dunnett's Test (Steel and Torrie, 1960). A significance level of p<0.05 was used. Cumulative mortality data were analysed using Fisher's Exact Probability Test, p<0.05 (Siegel, 1956). The pathologic data were statistically analysed using Fisher's Exact Probability Test and the Mantel-Haenzel Test (Siegel, 1956). A significance level of p<0.05 was used.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
The cumulative mortality for male rats exposed to 5.0 ppm HEA was statistically increased from equivalent data on control males in the 16th month of the study. This correlated with the onset of chronic murine pneumonia which initially affected this group and subsequently spread to the other exposure and control groups. During months 20-22, the males exposed to 5.0 ppm HEA had decreased cumulative mortality when compared to the male control group. The mortality of male rats exposed to 0.5 ppm HEA was comparable to the mortality of control males, except for a statistical increase in the 10th mouth and a statistical decrease in the 17th month of the study. The mortality of exposed female rats was comparable to mortality of control females except for a statistical increase in the 17th month for females exposed to 5.0 ppm HEA and a statistical increase In the 15th month for
females exposed to 0.5 ppm HEA. Overall, the cumulative mortality data did not suggest any unequivocal exposure-related effects with the possible
exception of initial increased mortality associated with the onset of chronic murine pneumonia in rats exposed to 5.0 ppm HEA.


BODY WEIGHT AND WEIGHT GAIN
Male rats assigned to groups exposed to 5.0 or 0.5 ppm HEA had statistically lower body weights than the controls prior to initiation of the exposure period. These lower body weights were also noted during the exposure period, but the rate of body weight gain was comparable for all groups of male rats. Body weights of female rats in the HEA exposure groups were also slightly lower than control rats at the onset of the study. These differences disappeared by the fifth day of the study. In female rats, sporadic differences in body weight between HEA exposed and control rats occurred throughout the study. Overall, there were no alterations of body weights at either sex that could be attributed to the exposure to either level of HEA.


HAEMATOLOGY
- 12-months interim examination:
The haematological values for the male rats exposed to HEA showed no differences from the values of control males. Haematologic data for female rats exposed to 5.0 ppm HEA were statistically higher in haemoglobin concentration and statistically lower in total white blood cell count than the similar values for control females. These statistical differences, which were not noted in females exposed to 0.5 ppm HEA, may or may not have been related to exposure to 5.0 ppm HEA.
- 5-months post-exposure examination:
There were no differences from controls, except for a statistical increase in red blood cell count for the male rats which had been exposed to 5.0 ppm HEA. These data indicate that if the statistically significant haematologic changes noted at the 12 -month examination were related to exposure to 5.0 ppm HEA, these changes were transient in nature and did not persist into the terminal phases of the study.


CLINICAL CHEMISTRY
There were no significant differences between control and exposed groups.


URINALYSIS
- 12-months interim examination:
Examination of the data on the male rats 4 days prior to the 12-month interim kill suggested a possible increase in occult blood from one rat in each exposed group. Subsequently the urinalysis was repeated at the time of the interim kill and showed no evidence of occult blood in any of the rats tested. All other parameters measured on urine of male rats showed no difference from the control group. The urine of female rats tested 4 days prior
to the interim kill showed a slight decrease in specific gravity when compared to that of the control group. Repetition of the urinanalysis at the time of the interim kill showed no apparent decrease of specific gravity when compared to control. All other parameters measured on urine of male rats showed no difference when compared to the control group.
- 5-months post-exposure examination:
None of the parameters measured showed differences between exposed and control groups for either sex.


ORGAN WEIGHTS
- 12-months interim examination:
There were no significant differences in weights of heart, liver, or kidneys for male rats exposed to HEA. However, there were significantly decreased terminal body weights for male rats exposed to 5 ppm or 0.5 ppm HEA. In addition, there was a significant increase in the brain/body weight ratio and in the testes/body weight ratio for male rats exposed to 0.5 ppm. These relative weight changes are considered secondary to the difference in total body weights. There were no significant differences in body weight or weights of brain, heart, liver or kidney for female rats exposed to HEA.
- Terminal sacrifice:
There were no statistically significant differences in body weights or weights of heart, liver, kidneys, or testes for either group of male rats. The male rats exposed to 0.5 ppm HEA had a borderline statistically lower mean brain weight than the control group. This borderline decrease was considered of no toxicologic significance. There were no statistically significant differences in body weights, or weights of brain, liver, or kidney for either group of HEA- exposed female rats. The female rats exposed to 5.0 ppm HEA did show a statistical decrease in the absolute weight of the heart compared to the control group. This observation was considered of no toxicologic significance in view of (1) lack of change in the relative weight of these hearts, and (2) the inclusion of one heart weight that was inordinately low in weight due to a lower total body weight.


GROSS PATHOLOGY / HISTOPATHOLOGY: NON-NEOPLASTIC
Gross and histopathologic examination of rats exposed to 5.0 ppm HEA revealed a characteristic yellow staining of the haircoat, as well as an increased incidence, increased severity and earlier onset of the lesions associated with chronic murine pneumonia. These exposure-related effects were not observed in rats exposed to 0.5 ppm HEA. (for more details see Remarks on results)


HISTOPATHOLOGY: NEOPLASTIC
Chronic exposure of rats to 5.0 or 0.5 ppm HEA did not cause an increase in any of the parameters used to assess carcinogenic potential.
(for more details see IUCLID5 Technical Dossier, Chapter 7.7)


Dose descriptor:
NOAEC
Effect level:
0.002 mg/L air (nominal)
Sex:
male/female
Basis for effect level:
other: Increased incidence, increased severity, and earlier onset of the lesions associated with chronic murine pneumonia at the high dose level.
Critical effects observed:
not specified

Details on Results Concerning Gross Pathology / Histopathology

 

Integument:

A statistically significant number of male and female rats exposed to 5.0 ppm HEA had a distinctive grossly-visible yellow staining of the haircoat that persisted into the post-exposure period of the study. This was considered to be the result of the contact of HEA with the haircoat. The yellow staining was not observed grossly on rats from the lower exposure level of HEA (0.5 ppm) . Microscopic examination of sections of the stained haircoat revealed no histologic alterations. An occasional rat had an epidermal inclusion cyst formation or neoplasm of the integument.

 

Respiratory System:

Chronic murine pneumonia occurred in the groups of rats used in this study. This was indicated by pulmonary consolidation, atelectasis, bronchiectasis or tenacious mucopurulent inflammation along the tracheobronchial system. Historically, the organism Mycoplasma has been cultured from lung-lesions of this type occurring in the testing laboratory. These cases of chronic murine pneumonia sometimes included abscess formation, pleuritis, pericarditis, rhinitis and/or tracheitis. In addition to the lesions of chronic murine pneumonia, all groups had rats with varying degrees of pulmonary inflammatory reactions or aggregates of alveolar macrophages, hematogenous pigment, cholesterol clefts or red blood cells.

Statistical evaluation:

An increase in the incidence of numerous gross or microscopically visible lesions occurring as part of or secondary to the chronic murine pneumonia in male and/or female rats exposed to 5.0 ppm HEA. This included pulmonary consolidation, congestion, bronchiectasis, peribronchiolar lymphoid hyperplasia, peribronchiolar fibrosis, focal purulent inflammation and epithelial hyperplasia of bronchi/bronchioles, diffuse epithelial hyperplasia of the trachea, pulmonary aggregates of haematogenous pigment, focal pulmonary atelectasis, and focal hypercellularity of alveolar walls secondary to aggregates of red blood cells within alveoli.

An increase in the incidence of pulmonary aggregates of haematogenous pigment and focal hypercellularity of alveolar walls secondary to aggregates of red blood cells within alveoli in lungs of female rats exposed to 0.5 ppm HEA.

An increase in the incidence of diffuse pulmonary congestion of female rats exposed to 0.5 ppm HEA.

Overall assessment of these data suggests exposure to 5.0 ppm HEA increased the severity of lesions occurring as part of chronic murine pneumonia. However, this appeared not to be the case with exposure of rats to 0.5 ppm HEA.

 

Lvmphoreticular System:

Inflammatory or hyperplastic changes occurred in some lymph nodes of some rats for all groups. Some lymph nodes contained increased amounts of haematogenous pigment, oedema, abscess formation or pooling of red blood cells. There were inflammatory reactions in thoracic lymph nodes secondary to chronic murine pneumonia.

Statistical evaluation:

Increases in the incidence of oedema, inflammation and reactive lymphoid hyperplasia of the thoracic lymph nodes of female rats exposed to 5.0 ppm HEA. Female rats exposed to 0.5 ppm HEA also had increased incidence of oedema and reactive lymphoid hyperplasia of thoracic lymph nodes. These inflammatory responses were considered to be associated with chronic murine pneumonia that occurred in these rats.

An increase in the incidence of oedema of mesenteric lymph nodes of female rats exposed to 5.0 ppm

 

Spleen:

Increased hemopoietic activity and haematogenous pigment was commonly observed in the spleen of some rats from both control and exposed groups. Reticuloendothelial hyperplasia and also splenic atrophy were noted in the spleens of some control and exposed rats.

 

Liver:

All groups, exposed and control, had rats with variable degrees of focal inflammation, necrosis, fatty metamorphosis, cytoplasmic vacuolization, swollen hepatocytes, bile duct hyperplasia, pericholangiolar inflammation, sinusoidal distention, aggregates of reticuloendothelial cells adjacent to degenerate hepatocytes, biliary cyst formation, and foci or areas of atypical hepatocytes. Extramedullary hematopoiesis or vascular distention also occurred in livers of rats from all groups.

Statistical analysis:

Decreased incidence of mottling and also congestion of the liver in male rats exposed to 5.0 ppm HEA.

Increased incidence of focal areas of swollen hepatocytes and focal aggregates of mononuclear cells in liver of male rats exposed to 5.0 ppm HEA.

An increase in the incidence of focal bile duct proliferation in livers of female rats exposed to 5.0 ppm HEA.

 

Thyroid and parathvroid glands:

Thyroid hyperplasia or adenoma formation was observed in rats of all control and exposed groups. This usually involved the interfollicular cells of the thyroid. The most frequent alteration in the parathyroid glands was hyperplasia occurring secondary to the age-related progressive chronic nephropathy.

 

Pancreas:

Focal atrophy of pancreatic acinar tissue and focal fibrosis was noted in both control and exposed groups of rats. Hyperplastic nodules and neoplasms of the pancreatic acini were also noted in both control and exposed groups. Some control and exposed rats had pancreatic islets that were enlarged, or neoplastic. A few rats had pancreatic islets showing slight cytoplasmic vacuolation.

 

Female Reproductive Svstem:

An age-related occurrence of uterine endometrial hyperplasia, polyp formation and ovarian cyst formation was observed. Various forms of uterine inflammation also occurred in rats of all groups. A few cases of uterine abscessation or cyst formation were noted. Neoplasm occurred in a few cases.

Statistical analysis:

An increase in the incidence of inflammation of the uterus in female rats exposed to 5.0 ppm HEA.

 

Male Reproductive System:

An age-related decrease in testicular spermatogenic activity was noted in rats of both control and exposed groups. This was sometimes accompanied by decreased content of spermatogenic cells in the epididymis. Also, the accessory sex glands were sometimes found to have a decreased content of secretory material.

Other infrequent observations included inflammation of the accessory sex glands, abscessation of preputial glands, or neoplasia.

Statistical evaluation:

An increased incidence of vascular fibrinoid degeneration in testes of male rats exposed to 5.0 ppm HEA.

 

Stomach:

All exposed and control groups had some rats with focal gastric erosions/ulcers, gastric hemorrhage or hyperemia. Dilatation of gastric pits was also noted upon microscopic examination of some rats from each of the exposed and control groups. Gastric mucosal hyperplasia or hyperkeratosis also occurred in a few control and exposed rats. Mineralization of the gastric wall was noted in some rats from each control and exposed group. These rats usually had chronic progressive nephropathy and uremia.

Statistical evaluation:

Increase in the incidence of microscopically visible dilatation of gastric pits in male rats exposed to 5.0 ppm HEA.

 

Small and large intestines:

Various inflammatory processes were noted in segments of the intestinal tract of some rats from all control and exposed groups. Isolated cases of diverticulum formation, focal ulceration, reactive lymphoid hyperplasia and neoplasia were also noted. Intestinal nematodiasis was also noted in some rats from all groups. Focal inflammation and/or necrosis of the mesenteric fat was noted in a few rats scattered amongst all exposed and control groups of rats.

 

Eyes:

All groups had rats with various inflammatory changes of the cornea or other components of the eye. Some rats had eyes with focal hyperplasia of the cornea epithelium.

 

Nervous System:

The most common lesions were hyperplastic or neoplastic proliferations of the pituitary gland; some of these were associated with haematogenous pigment aggregates, hemangiectasis, or compression of the adjacent portion of the brain. Some rats had vacuolar degeneration of the peripheral nerves, and other rats had neoplasms originating from the Schwann cells of the peripheral nerves.

 

Adrenal Glands:

Hyperplastic or neoplastic changes were observed in the adrenal cortex or medulla of rats from all groups. Hematocyst formation and vascular sinusoidal distention and cytoplasmic vacuolization of the adrenal cortical cells also occurred in all groups.

 

Cardiovascular System:

All groups, exposed and control, had rats with age-related myocardial degenerative changes, aortic and thoracic vessel mineralization, periarteritis of the mesenteric and other vessels (testes, liver, etc.). Thrombosis and hematoma formation were occasional complications of the mesenteric periarteritis. Thrombosis of the left atrium caused the death of some rats.

Statistical evaluation:

An increase in the incidence of a grossly-visible flaccidity of the myocardium of males exposed to 0.5 ppm HEA.

An increase in the incidence of a microscopically-visible degeneration of myocardial blood vessels in female rats exposed to 5.0 ppm HEA.

 

Urinary System:

An age-related progressive chronic nephropathy occurred in rats (especially males) from all groups of rats. This was sometimes accompanied by secondary parathyroid hyperplasia and mineralization of certain tissues, such as the gastric wall. Other incidental lesions in kidneys included inflammation or hyperplasia of renal pelvic epithelium, mineralized deposits, focal purulent inflammation, focal fibrosis, cyst formation, calculus formation or neoplasia. A few rats had diffuse or focal urocystitis, inflammation or hyperplasia of the urinary bladder mucosa.

 

Subcutaneous Tissues and Mammary Glands:

A substantial number of control and exposed females had evidence of mammary gland hyperplasia, neoplasia and/or galactocele formation. Epidermal inclusion cysts or subcutaneous or integumentary neoplasms were also noted in a few control and exposed rats.

Statistical evaluation:

Increase in the incidence of female rats having a total of 3 grossly-visible subcutaneous masses (suggestive of mammary tissue origin) in the groups exposed to 5.0 or 0.5 ppm HEA. However, this was not the case with female rats of either exposure group that had 1, 2, 4 or 5 subcutaneous masses suggestive of mammary tissue origin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
chronic
Experimental exposure time per week (hours/week):
30
Species:
rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: inhalation
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 May 1974 - 07 May 1976 (experimental)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well documented study report, acceptable for assessment with restrictions. In the study report, test parameters didn’t totally comply with the specific testing guideline, but were sufficient to accept data and study was conducted prior to the advent of GLP regulations (1982).
Principles of method if other than guideline:
Groups of 99-100 male and female rats were erposed to atmospheres containing 0 ppm (controls), 5.0 ppm (24 mg/m3) or 0.5 ppm (2.4 mg/m3) 2-hydroxyethyl acrylate (HEA) for 6 hours/day, 5 days/week over an 18 month period, and subsequently held for a post-exposure period of 5 months (males) and 6 months (females). The study included 12-month interim sacrifices for pathologic and cytogenetic examinations.
GLP compliance:
no
Remarks:
(Study was conducted prior to the advent of GLP regulations)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Spartan substrain
- Source: Spartan Research Animals, Michigan
- Housing: 3-4 animals/cage
- Diet: ad libitum
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): ambient
- Humidity (%): ambient
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 3.7 cubic meters stainless steel chambers under dynamic airflov- conditions
- System of generating vapours: The exposure atmosphere in each chamber was generated by metering liquid HEA at a calculated rate into the top of
a 15 inch glass column ( 2" diameter) that was heated to a temperature (ca. 80°C) hot enough to vaporize the HEA. Dry compressed air was introduced at the bottom of the glass column to sweep the vapours into the chamber where they were diluted with room air at a rate calculated to provide the desired HEA Concentration.


TEST ATMOSPHERE
- The nominal concentration of HEA in the chamber was calculated from the ratio of the amount of liquid HEA used to the rate of total chamber airflow.
- Brief description of analytical method used:
The concentration of HEA in each chamber was determined three or more times daily.
Analytical concentrations for the first 6 1/2 months of exposure were obtained by drawing 10 liters of air from the chamber at a rate of 1 liter/min through a charcoal tube. The HEA absorbed on the charcoal was extracted into 2 mL of carbon disulfide. The quantity of HEA in a 2 µL sample of carbon disulfide extract was analysed by gas chromatography (detector: FID).
For the last 11 1/2 months of the study HEA samples were collected by bubbling chamber air through water instead of charcoal. Improved reproducibility of sample analysis and convenience were the primary reasons for using water instead of charcoal. Fifty liters of air from the chamber were drawn through 20 mL of distilled water in a fritted glass bubbler at a rate of 1 liter/min. The quantity of HEA in a 2 µL sample of the trapping solution was analyzed by gas chromatography using the same conditions as before.
- Samples taken from breathing zone: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sixty per cent of the total exposure days for the low exposure level and 73 % of the total exposure days of the high exposure level were within 50 % of the an analytical concentrations. The high nominal concentration values and variability in analytical concentrations reflect the fact that HEA, having a relatively low vapour pressure, was difficult to vaporize. Much of the HEA dispensed into the vaporization apparatus was not vaporized, but that not vaporized was not subtracted from the amount dispensed in calculation of the nominal concentration. Although the analytical concentrations were not identical to the target concentrations, especially for the higher exposure level, the target concentrations of 0.5 and 5.0 ppm were referred to throughout the study report.
Duration of treatment / exposure:
18 months
Frequency of treatment:
6 hours per day, 5 days per week
Dose / conc.:
0.024 mg/L air (nominal)
Remarks:
corresponding to 5 ppm)
Dose / conc.:
0.002 mg/L air (nominal)
Remarks:
corresponding to 0.5)
No. of animals per sex per dose:
99 - 100
Control animals:
yes, sham-exposed
Details on study design:
- Post-exposure recovery period: Males: 5 months; females: 6 months
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: no details given


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: no details given


BODY WEIGHT: Yes
- Time schedule for examinations: no details given


HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to the scheduled 12-month interim sacrifice, and again after 5 months of the post-exposure observation period
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 rats/sex/exposure group
- Parameters were examined: packed cell volume (PVC), red blood cell count (RBC), hemoglobin concentration (Hgb), total white blood cell count (WBC) and differential white blood cell count.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of serum: prior to the scheduled 12-month interim sacrifice, and again after 5 months of the post-exposure observation period
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 5 and 9 or 10 rats/sex/exposure group, respectively
- Parameters were examined: blood urea nitrogen (DUN) concentration, serum alkaline phosphatase (AP) activity, and serum glutamic pyruvic transaminase (SGPT) activity


URINALYSIS: Yes
- Time schedule for collection of blood: prior to the scheduled 12-month interim sacrifice, and again after 5 months of the post-exposure observation period
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 5 and 10 rats/sex/exposure group, respectively
- Parameters were examined: Urinary specific gravity, pH, and the presence or absence of glucose, protein, ketones, bilirubin and occult blood were evaluated at both time intervals. Urinary urobilinogen was evaluated at the preterminal sampling interval only.



Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

An interim necropsy of 5 rats/sex/exposure group was conducted after 12 months exposure. Terminal necropsy was conducted after completion of
23 months (18 months exposure + 5 months post-exposure) for male rats, and after completion of 24 months (18 months exposure + 6 months post-exposure) for female rats.

The eyes of 5 rats/sex/exposure group were placed in Zenker's fixative. The trachea and lungs were removed as a unit and distended with formalin
fixative. The weights of the brain, heart, liver, kidneys and testes (males) were recorded for 5 rats/sex/group sacrificed after 12 months and also for 9-19 rats/sex/exposure group sacrificed at termination.

Gross examination:
Representative portions of the major organs and tissues, including brain, heart, liver, kidneys, testes, lungs, thoracic and/or mesenteric lymph nodes, salivary glands, pancreas, adrenals, spleen, thymus, aorta, skeletal muscle, small intestine, large intestine, thyroid gland, parathyroid glands, eyes (those not fixed in Zenker's fixative), esophagus, trachea, spinal cord, peripheral nerve, pituitary gland, epididymides, urinary bladder, accessory sex glands, adipose tissue, ovaries, uterus, nasal turbinates, and any gross lesion suggestive of an unexpected pathologic process or with a tumour formation was preserved in buffered 10 % formalin fixative. The eyes were examined with a glass slide technique and the data were entered with the gross data.

Microscopic examination:
- Control and High (5.0 ppm) Exposure Group: All available tissues from all rats were examined.
- Low (0.5 ppm) Exposure Group: In the absence of any discernible exposure-related lesions in the tissues from rats exposed to 5.0 ppm, the 12-month interim examination of rats of the 0.5 ppm exposure group was limited to grossly visible lesions suggestive of tumour formation. From the terminal necropsy, all available lungs, livers, kidneys, lymph nodes, tracheas, plus grossly visible lesions suggestive of an unexpected pathologic process
or tumour formation were examined from all rats.
Statistics:
Significance of differences between control and test values for hematology, clinical chemistry, body weights, organ weights, and organ/body weight ratio data was statistically determined by an analysis of variance and Dunnett's Test (Steel and Torrie, 1960). A significance level of p<0.05 was used. Cumulative mortality data were analysed using Fisher's Exact Probability Test, p<0.05 (Siegel, 1956). The pathologic data were statistically analysed using Fisher's Exact Probability Test and the Mantel-Haenzel Test (Siegel, 1956). A significance level of p<0.05 was used.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
The cumulative mortality for male rats exposed to 5.0 ppm HEA was statistically increased from equivalent data on control males in the 16th month of the study. This correlated with the onset of chronic murine pneumonia which initially affected this group and subsequently spread to the other exposure and control groups. During months 20-22, the males exposed to 5.0 ppm HEA had decreased cumulative mortality when compared to the male control group. The mortality of male rats exposed to 0.5 ppm HEA was comparable to the mortality of control males, except for a statistical increase in the 10th mouth and a statistical decrease in the 17th month of the study. The mortality of exposed female rats was comparable to mortality of control females except for a statistical increase in the 17th month for females exposed to 5.0 ppm HEA and a statistical increase In the 15th month for
females exposed to 0.5 ppm HEA. Overall, the cumulative mortality data did not suggest any unequivocal exposure-related effects with the possible
exception of initial increased mortality associated with the onset of chronic murine pneumonia in rats exposed to 5.0 ppm HEA.


BODY WEIGHT AND WEIGHT GAIN
Male rats assigned to groups exposed to 5.0 or 0.5 ppm HEA had statistically lower body weights than the controls prior to initiation of the exposure period. These lower body weights were also noted during the exposure period, but the rate of body weight gain was comparable for all groups of male rats. Body weights of female rats in the HEA exposure groups were also slightly lower than control rats at the onset of the study. These differences disappeared by the fifth day of the study. In female rats, sporadic differences in body weight between HEA exposed and control rats occurred throughout the study. Overall, there were no alterations of body weights at either sex that could be attributed to the exposure to either level of HEA.


HAEMATOLOGY
- 12-months interim examination:
The haematological values for the male rats exposed to HEA showed no differences from the values of control males. Haematologic data for female rats exposed to 5.0 ppm HEA were statistically higher in haemoglobin concentration and statistically lower in total white blood cell count than the similar values for control females. These statistical differences, which were not noted in females exposed to 0.5 ppm HEA, may or may not have been related to exposure to 5.0 ppm HEA.
- 5-months post-exposure examination:
There were no differences from controls, except for a statistical increase in red blood cell count for the male rats which had been exposed to 5.0 ppm HEA. These data indicate that if the statistically significant haematologic changes noted at the 12 -month examination were related to exposure to 5.0 ppm HEA, these changes were transient in nature and did not persist into the terminal phases of the study.


CLINICAL CHEMISTRY
There were no significant differences between control and exposed groups.


URINALYSIS
- 12-months interim examination:
Examination of the data on the male rats 4 days prior to the 12-month interim kill suggested a possible increase in occult blood from one rat in each exposed group. Subsequently the urinalysis was repeated at the time of the interim kill and showed no evidence of occult blood in any of the rats tested. All other parameters measured on urine of male rats showed no difference from the control group. The urine of female rats tested 4 days prior
to the interim kill showed a slight decrease in specific gravity when compared to that of the control group. Repetition of the urinanalysis at the time of the interim kill showed no apparent decrease of specific gravity when compared to control. All other parameters measured on urine of male rats showed no difference when compared to the control group.
- 5-months post-exposure examination:
None of the parameters measured showed differences between exposed and control groups for either sex.


ORGAN WEIGHTS
- 12-months interim examination:
There were no significant differences in weights of heart, liver, or kidneys for male rats exposed to HEA. However, there were significantly decreased terminal body weights for male rats exposed to 5 ppm or 0.5 ppm HEA. In addition, there was a significant increase in the brain/body weight ratio and in the testes/body weight ratio for male rats exposed to 0.5 ppm. These relative weight changes are considered secondary to the difference in total body weights. There were no significant differences in body weight or weights of brain, heart, liver or kidney for female rats exposed to HEA.
- Terminal sacrifice:
There were no statistically significant differences in body weights or weights of heart, liver, kidneys, or testes for either group of male rats. The male rats exposed to 0.5 ppm HEA had a borderline statistically lower mean brain weight than the control group. This borderline decrease was considered of no toxicologic significance. There were no statistically significant differences in body weights, or weights of brain, liver, or kidney for either group of HEA- exposed female rats. The female rats exposed to 5.0 ppm HEA did show a statistical decrease in the absolute weight of the heart compared to the control group. This observation was considered of no toxicologic significance in view of (1) lack of change in the relative weight of these hearts, and (2) the inclusion of one heart weight that was inordinately low in weight due to a lower total body weight.


GROSS PATHOLOGY / HISTOPATHOLOGY: NON-NEOPLASTIC
Gross and histopathologic examination of rats exposed to 5.0 ppm HEA revealed a characteristic yellow staining of the haircoat, as well as an increased incidence, increased severity and earlier onset of the lesions associated with chronic murine pneumonia. These exposure-related effects were not observed in rats exposed to 0.5 ppm HEA. (for more details see Remarks on results)


HISTOPATHOLOGY: NEOPLASTIC
Chronic exposure of rats to 5.0 or 0.5 ppm HEA did not cause an increase in any of the parameters used to assess carcinogenic potential.
(for more details see IUCLID5 Technical Dossier, Chapter 7.7)


Dose descriptor:
NOAEC
Effect level:
0.002 mg/L air (nominal)
Sex:
male/female
Basis for effect level:
other: Increased incidence, increased severity, and earlier onset of the lesions associated with chronic murine pneumonia at the high dose level.
Critical effects observed:
not specified

Details on Results Concerning Gross Pathology / Histopathology

 

Integument:

A statistically significant number of male and female rats exposed to 5.0 ppm HEA had a distinctive grossly-visible yellow staining of the haircoat that persisted into the post-exposure period of the study. This was considered to be the result of the contact of HEA with the haircoat. The yellow staining was not observed grossly on rats from the lower exposure level of HEA (0.5 ppm) . Microscopic examination of sections of the stained haircoat revealed no histologic alterations. An occasional rat had an epidermal inclusion cyst formation or neoplasm of the integument.

 

Respiratory System:

Chronic murine pneumonia occurred in the groups of rats used in this study. This was indicated by pulmonary consolidation, atelectasis, bronchiectasis or tenacious mucopurulent inflammation along the tracheobronchial system. Historically, the organism Mycoplasma has been cultured from lung-lesions of this type occurring in the testing laboratory. These cases of chronic murine pneumonia sometimes included abscess formation, pleuritis, pericarditis, rhinitis and/or tracheitis. In addition to the lesions of chronic murine pneumonia, all groups had rats with varying degrees of pulmonary inflammatory reactions or aggregates of alveolar macrophages, hematogenous pigment, cholesterol clefts or red blood cells.

Statistical evaluation:

An increase in the incidence of numerous gross or microscopically visible lesions occurring as part of or secondary to the chronic murine pneumonia in male and/or female rats exposed to 5.0 ppm HEA. This included pulmonary consolidation, congestion, bronchiectasis, peribronchiolar lymphoid hyperplasia, peribronchiolar fibrosis, focal purulent inflammation and epithelial hyperplasia of bronchi/bronchioles, diffuse epithelial hyperplasia of the trachea, pulmonary aggregates of haematogenous pigment, focal pulmonary atelectasis, and focal hypercellularity of alveolar walls secondary to aggregates of red blood cells within alveoli.

An increase in the incidence of pulmonary aggregates of haematogenous pigment and focal hypercellularity of alveolar walls secondary to aggregates of red blood cells within alveoli in lungs of female rats exposed to 0.5 ppm HEA.

An increase in the incidence of diffuse pulmonary congestion of female rats exposed to 0.5 ppm HEA.

Overall assessment of these data suggests exposure to 5.0 ppm HEA increased the severity of lesions occurring as part of chronic murine pneumonia. However, this appeared not to be the case with exposure of rats to 0.5 ppm HEA.

 

Lvmphoreticular System:

Inflammatory or hyperplastic changes occurred in some lymph nodes of some rats for all groups. Some lymph nodes contained increased amounts of haematogenous pigment, oedema, abscess formation or pooling of red blood cells. There were inflammatory reactions in thoracic lymph nodes secondary to chronic murine pneumonia.

Statistical evaluation:

Increases in the incidence of oedema, inflammation and reactive lymphoid hyperplasia of the thoracic lymph nodes of female rats exposed to 5.0 ppm HEA. Female rats exposed to 0.5 ppm HEA also had increased incidence of oedema and reactive lymphoid hyperplasia of thoracic lymph nodes. These inflammatory responses were considered to be associated with chronic murine pneumonia that occurred in these rats.

An increase in the incidence of oedema of mesenteric lymph nodes of female rats exposed to 5.0 ppm

 

Spleen:

Increased hemopoietic activity and haematogenous pigment was commonly observed in the spleen of some rats from both control and exposed groups. Reticuloendothelial hyperplasia and also splenic atrophy were noted in the spleens of some control and exposed rats.

 

Liver:

All groups, exposed and control, had rats with variable degrees of focal inflammation, necrosis, fatty metamorphosis, cytoplasmic vacuolization, swollen hepatocytes, bile duct hyperplasia, pericholangiolar inflammation, sinusoidal distention, aggregates of reticuloendothelial cells adjacent to degenerate hepatocytes, biliary cyst formation, and foci or areas of atypical hepatocytes. Extramedullary hematopoiesis or vascular distention also occurred in livers of rats from all groups.

Statistical analysis:

Decreased incidence of mottling and also congestion of the liver in male rats exposed to 5.0 ppm HEA.

Increased incidence of focal areas of swollen hepatocytes and focal aggregates of mononuclear cells in liver of male rats exposed to 5.0 ppm HEA.

An increase in the incidence of focal bile duct proliferation in livers of female rats exposed to 5.0 ppm HEA.

 

Thyroid and parathvroid glands:

Thyroid hyperplasia or adenoma formation was observed in rats of all control and exposed groups. This usually involved the interfollicular cells of the thyroid. The most frequent alteration in the parathyroid glands was hyperplasia occurring secondary to the age-related progressive chronic nephropathy.

 

Pancreas:

Focal atrophy of pancreatic acinar tissue and focal fibrosis was noted in both control and exposed groups of rats. Hyperplastic nodules and neoplasms of the pancreatic acini were also noted in both control and exposed groups. Some control and exposed rats had pancreatic islets that were enlarged, or neoplastic. A few rats had pancreatic islets showing slight cytoplasmic vacuolation.

 

Female Reproductive Svstem:

An age-related occurrence of uterine endometrial hyperplasia, polyp formation and ovarian cyst formation was observed. Various forms of uterine inflammation also occurred in rats of all groups. A few cases of uterine abscessation or cyst formation were noted. Neoplasm occurred in a few cases.

Statistical analysis:

An increase in the incidence of inflammation of the uterus in female rats exposed to 5.0 ppm HEA.

 

Male Reproductive System:

An age-related decrease in testicular spermatogenic activity was noted in rats of both control and exposed groups. This was sometimes accompanied by decreased content of spermatogenic cells in the epididymis. Also, the accessory sex glands were sometimes found to have a decreased content of secretory material.

Other infrequent observations included inflammation of the accessory sex glands, abscessation of preputial glands, or neoplasia.

Statistical evaluation:

An increased incidence of vascular fibrinoid degeneration in testes of male rats exposed to 5.0 ppm HEA.

 

Stomach:

All exposed and control groups had some rats with focal gastric erosions/ulcers, gastric hemorrhage or hyperemia. Dilatation of gastric pits was also noted upon microscopic examination of some rats from each of the exposed and control groups. Gastric mucosal hyperplasia or hyperkeratosis also occurred in a few control and exposed rats. Mineralization of the gastric wall was noted in some rats from each control and exposed group. These rats usually had chronic progressive nephropathy and uremia.

Statistical evaluation:

Increase in the incidence of microscopically visible dilatation of gastric pits in male rats exposed to 5.0 ppm HEA.

 

Small and large intestines:

Various inflammatory processes were noted in segments of the intestinal tract of some rats from all control and exposed groups. Isolated cases of diverticulum formation, focal ulceration, reactive lymphoid hyperplasia and neoplasia were also noted. Intestinal nematodiasis was also noted in some rats from all groups. Focal inflammation and/or necrosis of the mesenteric fat was noted in a few rats scattered amongst all exposed and control groups of rats.

 

Eyes:

All groups had rats with various inflammatory changes of the cornea or other components of the eye. Some rats had eyes with focal hyperplasia of the cornea epithelium.

 

Nervous System:

The most common lesions were hyperplastic or neoplastic proliferations of the pituitary gland; some of these were associated with haematogenous pigment aggregates, hemangiectasis, or compression of the adjacent portion of the brain. Some rats had vacuolar degeneration of the peripheral nerves, and other rats had neoplasms originating from the Schwann cells of the peripheral nerves.

 

Adrenal Glands:

Hyperplastic or neoplastic changes were observed in the adrenal cortex or medulla of rats from all groups. Hematocyst formation and vascular sinusoidal distention and cytoplasmic vacuolization of the adrenal cortical cells also occurred in all groups.

 

Cardiovascular System:

All groups, exposed and control, had rats with age-related myocardial degenerative changes, aortic and thoracic vessel mineralization, periarteritis of the mesenteric and other vessels (testes, liver, etc.). Thrombosis and hematoma formation were occasional complications of the mesenteric periarteritis. Thrombosis of the left atrium caused the death of some rats.

Statistical evaluation:

An increase in the incidence of a grossly-visible flaccidity of the myocardium of males exposed to 0.5 ppm HEA.

An increase in the incidence of a microscopically-visible degeneration of myocardial blood vessels in female rats exposed to 5.0 ppm HEA.

 

Urinary System:

An age-related progressive chronic nephropathy occurred in rats (especially males) from all groups of rats. This was sometimes accompanied by secondary parathyroid hyperplasia and mineralization of certain tissues, such as the gastric wall. Other incidental lesions in kidneys included inflammation or hyperplasia of renal pelvic epithelium, mineralized deposits, focal purulent inflammation, focal fibrosis, cyst formation, calculus formation or neoplasia. A few rats had diffuse or focal urocystitis, inflammation or hyperplasia of the urinary bladder mucosa.

 

Subcutaneous Tissues and Mammary Glands:

A substantial number of control and exposed females had evidence of mammary gland hyperplasia, neoplasia and/or galactocele formation. Epidermal inclusion cysts or subcutaneous or integumentary neoplasms were also noted in a few control and exposed rats.

Statistical evaluation:

Increase in the incidence of female rats having a total of 3 grossly-visible subcutaneous masses (suggestive of mammary tissue origin) in the groups exposed to 5.0 or 0.5 ppm HEA. However, this was not the case with female rats of either exposure group that had 1, 2, 4 or 5 subcutaneous masses suggestive of mammary tissue origin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
2.4 mg/m³
Study duration:
chronic
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity: Oral route

In a GLP-compliant oral 90-day repeated dose toxicity study according to OECD Guideline 408 the test substance was administered daily by oral gavage to 10 male and 10 female Wistar Han rats at doses of 0 mg/kg bw/day (test group 1), 10 mg/kg bw/day (test group 2), 30 mg/kg bw/day (test group 3) and 100 mg/kg bw/day (test group 4) for at least 90 consecutive days. The dose selection was based on the results of an OECD TG 422 in Wistar rats, in which strong irritation including erosion and ulcer in the forestomach, as well as inflammatory changes in the duodenum were detected in the parental animals of the high dose (150 mg/kg). The effects in the forestomach and duodenum were still observed in the mid dose (50 mg/kg), but less pronounced.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, functional observational battery, motor activity, ophthalmology, thyroid hormone assessment, clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis), spermatogenesis evaluation, gross necropsy findings, organ weights, and histopathologic examinations.

All animals survived to the scheduled necropsy. There were no test substance-related clinical observations or effects on body weight, hematology, coagulation, or urinalysis. There were no test substance-related ophthalmic, macroscopic, microscopic findings, or effects on ovarian follicle counts, thyriod hormones (T3, T4 or TSH levels), sperm morphology or differential counts.

Test substance-related higher food consumption was noted in the 100 mg/kg/day group males and females (Days 1–90), and in the 30 mg/kg/day group males (Days 29–90), compared to the control group.

Test substance-related higher cholesterol and potassium, and lower mean chloride levels were noted in the 100 mg/kg/day group males on Week 13, compared to the control group.

Test substance-related higher mean organ weights were noted in the liver and thyroid/parathyroid gland of the100 mg/kg/day group males. Higher mean kidney weights were present in the 100 mg/kg/day group males, and lower mean thymus weights were seen in the >10 mg/kg/day group females, but relationship of these to the test substance was considered uncertain.

Based on the results of this study, oral administration of the test substance to Wistar Han rats at dosage levels of 10, 30, and 100 mg/kg/day for a minimum of 90 days was well tolerated at all dosages. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 100 mg/kg/day.

Repeated dose toxicity: Inhalation route

An inhalation study with rats, mice, rabbits and dogs was conducted with hydroxypropyl acrylate (Dow Chemical Company 1983). Three groups of animals per species, consisting of 10 male Sprague-Dawley rats, 20 male Swiss-Webster mice, 4 male white rabbits, or 2 male beagle dogs, were used in the study. The animals were exposed (whole body) to hydroxypropyl acrylate vapours at 0 (unexposed control), 5 ppm (0.027 mg/L) or 10 ppm (0.053 mg/L). Exposures were 6 hours/day, 5 days/week for a total of 21 exposures for the rats and mice and 20 exposures for rabbits and dogs.

In the rat study, no mortality was observed. Five of 10 rats exposed to 10 ppm developed slight cloudiness of the cornea. No treatment-related decreases in mean body weights were found, and the absolute and relative organ weights were not affected. No treatment-related effects on the haematological measurements, the clinical chemistry measurements, or the urinalysis parameters were identified. At necropsy, focal corneal cloudiness was noted in 8 of 10 rats from the high exposure group suggesting an irritant effect. Histologically, a small increase in the number of animals with changes in the lungs at 10 ppm (subacute pneumonitis) and the nasal mucosa (both concentrations) was observed. No other treatment-related effects in the examined tissues were observed and no systemic toxicity was identified. The LOAEC was 5 ppm (0.027 mg/L) based on the effects in the nasal mucosa.

In the mouse study, no mortality was observed. Three of 20 mice exposed to 10 ppm, but none exposed to 5 ppm showed signs of eye irritation. No compound-related, statistically significant effects on mean body weights were found. However, the high dose mice showed slightly lower body weights during weeks 1, 3 and 4 of exposure (up to 4.5 % lower in week 1) which recovered during the weekend without treatment. No gross or histopathological changes related to exposure were observed. The NOAEC was 5 ppm (0.027 mg/L).

In the rabbit study, no mortality was observed. All rabbits exposed to 10 ppm and 2 of 4 animals exposed to 5 ppm developed slight rhinitis and eye irritation (moderate conjunctivitis). No treatment-related effects on mean body weight were observed and all groups gained a similar amount of weight during the study. The absolute and relative organ weights were not affected. No treatment-related effects on the haematological measurements, or the clinical chemistry measurements were identified. Gross and histopathological changes related to exposure were found in the upper respiratory system (rhinitis, squamous metaplasia, and ulcerations), trachea and lungs (bronchitis, focal pneumonitis) of both hydroxypropyl acrylate exposure groups. The tracheal changes varied from no effect in some low dose animals to focal ulceration, squamous metaplasia and tracheitis in the high dose group. The most marked effects were seen in the upper respiratory system of all rabbits, especially those in the 10 ppm group that were characterized by mucopurulent rhinitis with squamous metaplasia and ulceration of the nasal turbinate mucosa. The LOAEC was 5 ppm (0.027 mg/L) based on effects on the upper respiratory system.

In the dog study, no mortality was observed. During the exposures, both dogs exposed to 10 ppm lost body weight (from 2 to 10 %) and exhibited nasal irritation (exudative rhinitis) and eye irritation (bilateral corneal cloudiness, slight corneal edema, and bilateral suppurative conjunctivitis). The body weights showed some recovery during the weekends when no exposures occurred. One dog exposed to 5 ppm exhibited exudative rhinitis approximately half way through the study. At necropsy, exudative rhinitis, tracheitis, and suppurative bronchopneumonia were observed in all hydroxypropyl acrylate-exposed dogs. Microscopic lesions included suppurative rhinitis, squamous metaplasia and hyperplasia of the lining epithelium and focal area of ulceration in the mucosa of the nasal turbinates. These lesions extended to the trachea and lungs of the high exposure group resulting in bronchopneumonia. The LOAEC was 5 ppm (0.027 mg/L) based on effects on the upper respiratory system.

In addition, there are data from the structural analogue 2-hydroxyethyl acrylate available:

In a 4-week inhalation study 15 to 20 male Sherman rats per group were exposed for 7 hours/day, 5 days/week to HEA vapours at concentrations of 0, 5, 10 or 25 ppm (corresponding to approx. 0.024, 0.048, and 0.120 mg/L) (Dow Chem. Co., 1970).Mean analytical concentrations: 4.5 ± 1.1 ppm, 10.6 ± 1.4 ppm, and 22.5 ± 3.9 ppm. Theduration of exposures was seven hours per day, five days per week for a total of twenty exposures. Interim sacrifices were performed on the 5 and 10 ppm groups after 2 weeks of exposure and on the 25 ppm animals after one week of exposure. All animals were subjected to a gross and microscopic examination irrespective of whether they died during treatment or were sacrificed at termination of treatment. Clinical signs of mild nasal irritation were observed at 10 ppm; concentrations of 25 ppm produced dyspnoea and abdominal bloating which became more severe as the number of exposures increased.

Histopathological examination found ulcerative keratitis (superficial loss of cornea with inflammation) in all groups exposed to HEA. Ulcerative keratitis appeared to be an HEA dose-related lesion with lesions in 14, 6, and 3 animals at levels of 25 ppm, 10 ppm and 5 ppm, respectively. Twenty exposures produced a higher incidence of corneal ulcers than 8 or 9 exposures at the 10 ppm and 5 ppm level. Focal ulcerative rhinitis (superficial loss of nasal epithelial tissue with inflammation) was observed in 7 and 4 rats in the 25 and 10 ppm exposure groups, respectively. This lesion was not present in the 14 days recovery rats on the above doses or in any 5 ppm and control rats.The higher incidence of chronic-active laryngitis in the HEA-exposed rats was interpreted as being compound-related at the 25 ppm and 10 ppm levels as was the chronic-active tracheitis at the 25 ppm, 10 ppm, and 5 ppm levels.There were 17 spontaneous deaths in the 25 ppm treatment group. Unfortunately due to the high incidence of chronic murine pneumonia in all groups (not treatment-related) it was impossible to characterize any lung pathology which might have been caused by exposure to HEA. At termination, mean body weights of rats exposed to 10 ppm for 20 days were significantly lower than controls. Relative weights of livers were higher for rats that were exposed to 10 and 5 ppm, relative kidney weight was increased at 10 ppm only. Testicular atrophy was observed histopathologically in one of 9 rats exposed to 10 ppm HEA for 20 exposures but was judged not to be treatment-related. No testicular atrophy was found in the highest exposure group. The lowest observed adverse effect concentration (LOAEC), based on severe local irritation effects (ulcerative keratitis and chronic-active tracheitis), was 5 ppm. No NOAEC was derived in this study.

In a chronic inhalation study male and female Sprague-Dawley rats (99 or 100 animals/ sex/dose group) were exposed to atmospheres containing 0 ppm (controls), 5.0 ppm (0.024 mg/L) or 0.5 ppm (0.0024 mg/L) 2-hydroxyethyl acrylate (HEA) for 6 hours/day, 5 days/week over an 18 month period, and subsequently held for a post-exposure period of 5 months (males) and 6 months (females). The study included 12-month interim sacrifices for pathologic and cytogenetic examinations. Haematology, blood chemistry and urine analysis were measured prior to the scheduled 12-month interim sacrifice, and again after 5 months of the post-exposure observation period. Histopathological examination was carried out for the all available tissues of the control and 5 ppm groups at interim and terminal sacrifice, and at 0.5 ppm terminal sacrifice limited evaluations were performed.

Body weights, terminal organ weights and cumulative mortality, urinalysis, clinical chemistry and haematology did not appear to be altered by chronic HEA exposure. Overall treatment was not associated with adverse effects except that the rats in the 5 ppm treatment group developed yellow staining of the fur and a marginal increase in Mycoplasma-induced chronic murine pneumonia which was interpreted as being treatment-related. No treatment-related effects were seen in the 0.5 ppm group.

Overall chronic inhalation exposure to HEA at a dose of 5 ppm caused only a minimal toxicological effect while no toxicity was seen at 0.5 ppm. Gross and histopathological examination of tissues showed no indication of significant chronic toxicity or a carcinogenic effect in either the 5 or 0.5 ppm treatment groups (Dow Chemical Co., 1979). The NOAEC was set at 0.0024 mg/L (nominal) based on an increased incidence, increased severity, and earlier onset of the lesions associated with the chronic murine pneumonia observed at the high dose level.

Repeated dose toxicity: Dermal route

There are no experimental data available on subchronic and chronic toxicity of hydroxypropyl acrylate by the dermal route of exposure. But due to animal welfare reasons and since the substance is corrosive and moderate to strong skin sensitizer, no long-term testing by the dermal route is planned.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.