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Administrative data

Description of key information

2 -hydroxyethyl acrylate (HEA) itself is comprehensively tested in respect to subacute (rat inhalation), subchronic (rat oral, dog oral) and chronic toxicity (rat inhalation). The data are supported by tests performed with the structural analogue 2 hydroxypropyl acrylate (HPA) which was tested in subacute studies in rat, mouse, dog and rabbit by inhalation exposure and in subchronic studies in rats after oral administration.

Inhalation

In the 2 years inhalation study with male/female Sprague-Dawley rats (DowChemCo, 1979) the lowest observed adverse effect concentration (LOAEC), based on severe local irritation effects was 5 ppm (equivalent to approx. 0.024 mg/L). No toxicity was observed in rats exposed to 0.5 ppm HEA (corresponding to 0.0024 mg/L), thus NOAEC in this study was determined to be 0.0024 mg/L. 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.

Oral

In the subchronic oral feed studies in rat and dogs no systemic toxicity were described up to the highest dose tested (rat: males 196 mg/kg bw/day; females 305 mg/kg bw/day) (dog: males 125 mg/kg bw/day; females 131 mg/kg bw/day).

In a study according to OECD TG 422, the oral administration of HEA by gavage to male and female Wistar rats resulted in signs of parental toxicity at the mid- and high-dose of 40 and 120 mg/kg bw/d, such as a combination of clinical signs and gastrointestinal pathology, being the consequence of the irritating properties of the test item. 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.

The structural analog hydroxypropyl acrylate (HPA) was also tested in an 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. The NOAEL for fertility and reproductive performance was 150 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the F1 progeny was 150 mg/kg bw/d.

In a study according OECD TG 408 performed with the structurally analogue substance HPA the NOAEL for systemic toxicity was 100 mg/kg bw/day. This study is used in a read across approach for HEA.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Study was conducted prior to the advent of GLP regulations (1982)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
Deviations:
yes
Remarks:
lesser haematological and clinical parameters have been investigated and no urinalysis was conducted; only 2 animals/dose/sex tested
Principles of method if other than guideline:
Groups of male and female Beagle dogs (2/sex/group) were maintained for 97 days on a diet containing 0.06, 0.2, or 0.4 % HEA in diet (equivalent to doses of 21, 60 and 125 and 22, 63 and 131 mg/kg body weight/day for males and females respectively).
GLP compliance:
no
Remarks:
(Study was conducted prior to the advent of GLP regulations)
Limit test:
no
Specific details on test material used for the study:
- Analytical purity: 97 %
- Source: Texas Division
- Physical state: pale yellow liquid
- Density: ca. 1.07
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6-7 months
- Weight at study initiation: no data
- Housing: 2 dogs/cage
- Diet (ad libitum): Purina Laboratory Chow mixed with 1 % peanut oil
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
no details
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
97 days
Frequency of treatment:
daily
Dose / conc.:
21 mg/kg bw/day (nominal)
Remarks:
administered in males
Dose / conc.:
60 mg/kg bw/day (nominal)
Remarks:
administered in males
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
administered in males
Dose / conc.:
22 mg/kg bw/day (nominal)
Remarks:
administered in females
Dose / conc.:
63 mg/kg bw/day (nominal)
Remarks:
administered in females
Dose / conc.:
131 mg/kg bw/day (nominal)
Remarks:
administered in females
No. of animals per sex per dose:
2
Control animals:
yes, plain diet
Details on study design:
Post-exposure period: no
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: frequently

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: frequently

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Food consumption records were kept weekly throughout the experimental period

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before test start and after 83 days on the diet
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all
- Parameters examined: hematocrit, RBC, hemoglobin, WBC, differential count

CLINICAL CHEMISTRY: Yes
- How many animals: all
- Parameters examined: serum urea nitrogen, alkaline phosphatase, bromsulfophthalein (B.S.P.), serum glutamic oxalacetic transaminase (S.G.O.T.), and serum glutamic pyruvic transaminase (S.G.P.T.) values

URINALYSIS: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

All animals were fasted overnight and weighed before examination at autopsy. Bone marrow smears were taken from the rib of the male and female dogs on the control and 0.4 percent levels of 2-hydroxyethyl acrylate and from the female dogs on the control. The lungs, heart, liver, kidneys, spleen, brain, and testes were removed and weighed.
Portions of each organ, as well as lymph node, esophagus, aorta, pancreas, uterus, ovary, urinary bladder, gall bladder, stomach, skeletal muscle, large intestine, small intestine, spinal cord, pituitary gland, adrenal gland, thyroid, parathyroid, and peripheral nerve were preserved. Hematoxylin-eosin stained sections of the tissues were prepared for microscopic examination.
Statistics:
no data
Clinical signs:
no effects observed
Description (incidence and severity):
No compound-related effects
Mortality:
no mortality observed
Description (incidence):
No compound-related effects
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Further details / tables see attachments.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No compound-related effects

Further details / tables see attachments.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No compound-related effects

Further details / tables see attachments.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No compound-related effects

Further details / tables see attachments.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No compound-related effects

Further details / tables see attachments.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No compound-related effects

Further details / tables see attachments.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No compound-related effects

Further details / tables see attachments.
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: high dose
Key result
Dose descriptor:
NOAEL
Effect level:
131 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: high dose
Key result
Critical effects observed:
no

One female dog on the 0.4 percent level of 2 -hydroxyethyl acrylate was relatively small at the beginning of the experimental period and showed a slight weight loss during this period. This dog was found to have an enlarged spleen. However, upon microscopic examination of the tissues from this animal, no evidence of adverse changes was noted. Thus, the dog's condition was not attributed to the experimental compound.

 

Thus, there was no evidence of adverse effects as judged by general appearance and behavior, weight gain, food consumption, hematological values, examination of bone marrow, urea nitrogen content and alkaline phosphatase activity in the serum, serum bromsulfophthalein, serum glutamic oxalacetic transaminase and serum glutamic pyruvic transaminase tests, final average body and organ weights, and gross and microscopic examination of tissues.

Details on hematology, clinical chemistry, body weight, organ weights and pathological/microscopic examinations are given in the attachment.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
lesser parameters have been investigated like only two clinical parameters and no haematology parameters for the test groups have been reported; no urinalysis was conducted
Principles of method if other than guideline:
Groups of male and female Sherman strain rats (10/sex/group) were maintained for 100 days on a diet containing 0, 0.03. 0.1 or 0.3 % HEA (equivalent to doses of approx. 0, 20, 65, and 196 mg/kg body weight/day for males and 0, 30, 102, and 305 mg/kg body weight/day for females). The feeding study was performed pre-OECD TG and pre-GLP, but the study is well reported using an adequate number of animals (comparable to current OECD TG 408) pathological and histopathological examinations were performed and the results reported.
GLP compliance:
no
Remarks:
(Study was conducted prior to the advent of GLP regulations)
Limit test:
no
Specific details on test material used for the study:
- Analytical purity: 97 %
- Source: Texas Division
- Physical state: pale yellow liquid
- Density: ca. 1.07
Species:
rat
Strain:
Sherman
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 64 days
- Weight at study initiation: no data
- Housing: 2 rats/cage
- Diet: ad libitum, Purina Lboratory Chow
- Water: ad libitum
- Acclimation period: since weaning at the age of 21 days

ENVIRONMENTAL CONDITIONS
no details
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
100 days
Frequency of treatment:
daily
Dose / conc.:
0.03 other: % nominal in diet
Remarks:
equivalent to doses of approx. 20 and 30 mg/kg bw /day for males and females, respectively
Dose / conc.:
0.1 other: % nominal in diet
Remarks:
equivalent to doses of approx. 65 and 102 mg/kg bw /day for males and females, respectively
Dose / conc.:
0.3 other: % nominal in diet
Remarks:
equivalent to doses of approx. 196 and 305 mg/kg bw /day for males and females, respectively
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
Post-exposure period: no

Animals were weighed twice weekly for the first 28 days, and once a week thereafter. They were observed frequently for gross changes in appearance or behavior. Records were kept of mortality and food consumption was recorded for the first month. Terminal hematological values were obtained from 5 male and 5 female rats for the control group. Samples of blood serum were obtained for the determination of urea nitrogen content and alkaline phosphatase activity.

At autopsy animals were fasted overnight, weighed and killed by decapitation. The lungs, heart, liver, kidneys, spleen, testes and brain were removed examined and weighed. Portions of these organs, as well as pituitary, thyroid, parathyroid, adrenal, pancreas, lymph nodes, peripheral nerve, urinary bladder, esophagus, stomach, small intestine, large intestine, ovary and uterus were preserved in 10% neutral formalin and hematoxylin-eosin stained sections were prepared for histological examinations.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: frequently

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: frequently

BODY WEIGHT: Yes
- Time schedule for examinations: during the first 28 days of the study twice weekly, then once a week thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
- Time schedule for food consumption: recorded during the first 30 days

HAEMATOLOGY: no

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before sacrifice
- Animals fasted: Yes
- Parameters: urea nitrogen content and alkaline phosphatase activity

URINALYSIS: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

At autopsy the animals were fasted overnight, weighed, and sacrificed by decapitation. The lungs, heart, liver, kidneys, spleen, testes, and brain were removed, examined, and weighed. Portions of these organs, as well as pituitary, thyroid, parathyroid, adrenal, pancreas, lymph node, peripheral nerve, urinary bladder, esophagus, stomach, small intestine, large intestine, ovary, and uterus were preserved in 10 % neutral formalin and hematoxylin-eosin stained sections were prepared for histological examination.
Statistics:
When appropriate, the Fisher "t"-test was used in comparing the mean values obtained on the experiment groups with those of the controls; in general, probability values (P) of less than 0.05 were interpreted as indicating a significant difference.
Clinical signs:
no effects observed
Description (incidence and severity):
No substance-related effects.
Mortality:
no mortality observed
Description (incidence):
No substance-related effects.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No substance-related effects.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No substance-related effects.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No substance-related effects.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No substance-related effects.

Further details / tables see attachments.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No substance-related effects.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No substance-related effects.

Further details / tables see attachments.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No substance-related effects.

Further details / tables see attachments.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
>= 196 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: high dose
Key result
Dose descriptor:
NOAEL
Effect level:
>= 305 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: high dose
Key result
Critical effects observed:
no

The rats were maintained for 100 days on diets containing HEA without evidence of adverse effect as judged by general appearance and behaviour, growth, food consumption, serum, urea, nitrogen and alkaline phosphatase determinations, final average body and organ weights, organ/body weight ratios, and gross and microscopic examination of tissues. Gross examination of all tissues was made at autopsy and microscopic examination of hematoxylin and eosin stained sections was made of brain, pituitary, thyroid, parathyroid, adrenals, pancreas, spleen, lymph node, lung, heart, liver, kidney urinary bladder, esophagus, stomach, small intestine, large intestin, and gonads.

See attachment for body weights, organ weights and pathological observation.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Sep 2019 - July 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 Jul 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: PAU 0118813

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator (KS)
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 13-14 weeks (male) / about 13 weeks (female)
- Weight at study initiation: mean males= 374 g, mean females=212 g
- Fasting period before study: no
- Housing: Polysulfonate cages: -During pretreatment: up to 5 animals per sex and cage; -During premating: 2 animals per sex and cage; -During mating and postmating: 2 animals per cage (males only)
Polycarbonate cages: 1 animal ; Exceptions: -During overnight mating: 1 male/1 female per cage; -During rearing up to PND 13: 1 dam with her litter
- Diet: ad libitum (Mouse and rat maintenance diet “GLP”, Granovit AG, Kaiseraugst, Switzerland)
- Water: ad libitum (drinking water)
- Acclimation period: 3 weeks

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

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance solutions in drinking water were prepared in intervals, which took into account the analytical results of the stability verification. For the preparation of the administration solutions the test substance was weighed in a graduated flask depending on the dose group, topped up with ultrapure water and intensely mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.
The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All measured values for the test item were in the expected range of the target concentrations (90 - 110%) demonstrating the correctness of the preparations. The stability of test substance in drinking water was demonstrated for a period of 7 days at room temperature.
Duration of treatment / exposure:
The duration of treatment covered a 5 weeks in-life period (males) including 14 days mating (mating pairs were from the same test group) as well as a 2-weeks premating period (females), 14 days mating period, 9 days postmating period in one female (for no evidence of sperm), the entire gestation and about 3 weeks of lactation period in females up to one day prior to the day of scheduled sacrifice of the animals.
Frequency of treatment:
once daily
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
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- A cageside examination was conducted at least daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration

DETAILED CLINICAL OBSERVATIONS: Yes
- 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. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.
Detailed clinical observations (DCO) were performed in all animals once prior to the first administration (day 0) and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable. For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 × 37.5 × 25 cm). The following parameters listed were assessed:
Abnormal behavior in “handling”, Fur, Skin, Posture, Salivation, Respiration, Activity/arousal level, Tremors, Convulsions, Abnormal movements, Gait abnormalities, Lacrimation, Palpebral closure, Exophthalmos (Protruding eyeball), Assessment of the feces excreted during the examination (appearance/consistency), Assessment of the urine excreted during the examination, Pupil size

BODY WEIGHT: Yes
In general, the body weight of the male and female parental animals was determined 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 evidence 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 and 13.
Females without positive evidence of sperm, without litter and females after weaning (PND 13) were weighed weekly. These body weight data were solely used for the calculations of the dose volume; therefore these values are not reported in the Summary but in the Individual Tables.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Generally, food consumption was determined once a week (over a period of 7 days) for male and female parental animals, with the following exceptions:
Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (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 and 10 - 13.
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.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Generally, water consumption was determined once a week as representative value over a period of 3 days for the male and female parental animals, with the following exceptions:
Water consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (female parental animals)
Water consumption of the females with evidence of sperm was determined on gestation days (GD) 0-1, 6-7, 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 and 12-13.
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
- Time schedule for collection of blood: end of treatment
- Anaesthetic used for blood collection: Yes /isollurane
- Animals fasted: Yes
- How many animals: all
- Parameters checked: 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 (PLT), Differential blood count, Reticulocytes (RETA); Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument, Clotting tests were carried out using a ball coagulometer

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:end of treatment
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase; EC 2.6.1.2.), Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase; EC 2.6.1.1.), Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase; EC 3.1.3.1.), gamma-Glutamyltransferase (GGT) (gamma -glutamyl) peptide: aminoacid--glutamyl-transferase; EC 2.3.2.2.), sodium, Potassium, Chloride, Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total proteine, Albumine, Globulins, Triglycerides, Cholesterol, Bile acids

URINALYSIS: No

IMMUNOLOGY: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- A functional observational battery (FOB) was performed in the first 5 male and the first 5 female animals with litter per group (in order of delivery) at the end of the administration period starting at about 10.00 h. The FOB started in a randomized sequence.
The already single-housed female animals were transferred to new cages before the test, nor was drinking water withdrawn. At least 30 minutes before the start of the FOB the male animals were transferred from group housing to single animal polycarbonate cages type III, for the duration of the test. Drinking water was provided ad libitum, but no food was offered during the measurements. Likewise, food was withdrawn from the female animals for the duration of the test.
The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
The animals were observed in their closed home cages (for a short period: about 10-30 seconds); any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Gait, Other findings
The animals were transferred to a standard arena (50 × 50 × 25 cm) and observed. The following parameters were examined: Behavior on removal from cage, Fur, Skin, Salivation, Nasal discharge, Lacrimation, Eyes/pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/stereotypy, Gait, Activity/arousal level, Feces (appearance/ consistency) within 2 minutes, Urine (amount/color) within 2 minutes, Rearing within 2 minutes, Other findings

Sensory motor tests/Reflexes
The animals were removed from the open field and subjected to following sensory motor or reflex tests: Reaction to an object being moved towards the face (Approach response), Touch sensitivity (Touch response), Vision (Visual placing response), Pupillary reflex, Pinna reflex, Audition (Startle response), Coordination of movements (Righting response), Behavior during handling, Vocalization, Pain perception (Tail pinch), Other findings, Grip strength of forelimbs, Grip strength of hindlimbs, Landing foot-splay test

Motor activity measurement
The Measurement of motor activity (MA) was measured at the end of the administration period in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group. Motor activity (MA) was measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts were counted over 12 intervals for 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and was finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

Weight parameters: The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (final body weight); Epididymides; Ovaries; Prostate (ventral and dorsolateral part together, fixed); Seminal vesicles with coagulating glands (fixed); Testes; Thyroid glands (with parathyroid glands) (fixed); Uterus with cervix
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands (fixed); Brain; Heart; Kidneys; Liver; Spleen; Thymus (fixed)
Organ / Tissue fixation:
The following organs or tissues of all parental animals are fixed in 4% neutral buffered formaldehyde solution or modified Davidson’s solution: All gross lesions; Adrenal glands; Aorta; Bone marrow (femur); Brain; Cecum; Cervix; Coagulating glands; Colon; Duodenum; Esophagus; Extraorbital lacrimal glands; Epididymides (modified Davidson’s solution); Eyes with optic nerve (modified Davidson’s solution); Femur with knee joint; Heart; Ileum; Jejunum (with Peyer’s patches); Kidneys; Larynx; Liver; Lungs; Lymph nodes (axillary and mesenteric); Mammary gland (male and female); Nose (nasal cavity); Ovaries (modified Davidson’s solution); Oviducts; Pancreas; Parathyroid glands; Pharynx; Pituitary gland; Prostate; Rectum; Salivary glands (mandibular and sublingual); Sciatic nerve; Seminal vesicles; Skeletal muscle; Spinal cord (cervical, thoracic and lumbar cord); Spleen; Sternum with marrow; Stomach (forestomach and glandular stomach); Target organs; Testes (modified Davidson’s solution); Thymus; Thyroid glands; Trachea; Urinary bladder; Uterus; Vagina

HISTOPATHOLOGY: Yes
All gross lesions; Adrenal glands; Brain; Cecum; Cervix; Coagulating glands; Colon; Duodenum; Epididymides; Eyes with optic nerve; Heart; Ileum; Jejunum; Kidneys; Liver; Lungs; Lymph nodes (axillary and mesenteric); Ovaries; Oviducts; Prostate; Peyer’s patches; Rectum; Sciatic nerve; Seminal vesicles; Skeletal muscle; Spinal cord (cervical, thoracic, lumbar); Spleen; Sternum with marrow; Stomach (forestomach and glandular stomach); Testes; Thymus; Thyroid glands; Trachea; Urinary bladder; Uterus; Vagina
Other examinations:
Blood samples from the adult males were assessed for serum levels for thyroid hormones (T4 and TSH). The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter. T4 Elisa was measured with a Sunrise MTP-reader.

For all females in a pool of up to 50 animals, estrous cycle normality was evaluated before the randomization (the estrous cycle data of these individuals were not reported and can be found in the raw data). For a minimum of 2 weeks prior to mating estrous cycle length was evaluated by daily analysis of vaginal smear forall 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.

Parameters examined in F1 male parental generations:
testis weight, epididymis weight, stages of spermatogenesis in the testes and histopathology of interstitial testicular cell structures

Statistics:
Weight of the anesthetized animals and absolute and relative organ weights: KRUSKAL-WALLIS H and WILCOXON test
Clinical pathology parameters: KRUSKAL-WALLIS and WILCOXON-Test
Food consumption (parental animals), water consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days, anogenital distance, anogenital index: DUNNETT test (two-sided)
Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups: FISHER'S EXACT test (one-sided)
Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development: WILCOXON test (one-sided+) with BONFERRONI-HOLM
Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, survival index: WILCOXON test (one-sided-) with BONFERRONI-HOLM
% live male day x, %live female day x: WILCOXON test (two-sided)
Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity: KRUSKAL-WALLIS and WILCOXON test (two-sided)
Number of cycles and Cycle Length: KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All mid-and high-dose males showed salivation during the entire study. Salivation was also observed in most of the high-dose females during the premating, mating and gestation period, in all high-dose females during the lactation period, in one mid-dose female during the premating period and in some mid-dose females during the gestation and lactation period.
The temporary salivation was considered to be test substance-induced. Salivation occurred immediately after dosing (up to 2 hours post dosing) in the mentioned animals during the treatment period. It is likely, that this temporary finding was induced by a local affection of the upper digestive tract.
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 (1 - 3; 12, 40 and 120 mg/kg bw/d) during the study.
One mid-dose male (No. 30) showed piloerection on study day 21. One high-dose male (No. 32) had an injury of his hindlimbs (toe) during study days 30 - 33. One mid-dose female (No. 122) had an injury of the anogenital region during premating days 2 – 7.
Two high-dose females (Nos. 132 and 138) did not nurse their pups properly during PND 1- 3 and PND 1 - 2, respectively. As a consequence, several pups of high-dose female No. 132 and the only pup of high-dose female No. 138 died within 3 days after they were born.

Further detailed tables see attachment
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of all male and female parental animals and body weight change of all female parental animals in all test substance-treated groups were comparable to the concurrent control during the entire study period. Body weight gain was lower in all treated groups at the beginning of exposure (though statistically significant only in the high-dose parental males during study days 0 – 7), suggesting that the animals needed a few days to get adapted to the bolus gavage of this irritating compound. After this adaptation the mean body weight change was generally comparable to the concurrent control values during the rest of the study. The statistically significantly higher mean body weight change in the mid-dose males during study days 13 - 21 was considered to be spontaneous in nature and not treatment related since it was not related to dose.

Further detailed tables see attachment
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the high-dose male animals was statistically significantly above the concurrent control values during study days 7 - 13 and 0 - 13 (about 42% and 21%, respectively). The most likely explanation for this is that the animals spilled food.
Food consumption of the high-dose female animals was below control throughout lactation, the difference became statistically significant during PND 7 - 10 (about 23%). Overall the high-dose females consumed about 14% less food than the control females during lactation. Food consumption of the low- and mid-dose males and females during the entire study, and of the high-dose females during the premating and gestation period was comparable to the concurrent control values.

Further detailed tables see attachment
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Water consumption of all male animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study. Water consumption of the mid-dose females during the entire study, and of the low- and high-dose females during the premating and gestation period was comparable to the concurrent control values. Water consumption of the high-dose and low-dose females was statistically significantly below the concurrent control values during PND 6 - 7 (about 17% and 20%, respectively). As there was no dose-response no association to the treatment was assumed.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed. At the end of the administration period, in males of test groups 1, 2 and 3 (12, 40 and 120 mg/kg bw/d) relative eosinophil counts were decreased (in test group 2 not statistically significantly). The same was true for significantly decreased mean corpuscular hemoglobin concentration (MCHC) in females of test group 3 (120 mg/kg bw/d). However, the values were within historical control ranges (males, relative eosinophils 1.3-2.7 %; females, MCHC 20.84-22.23 mmol/L). In females of test group 2 (40 mg/kg bw/d) total white blood cell (WBC) counts and absolute neutrophil counts were significantly decreased. In females of test group 1 (12 mg/kg bw/d) absolute neutrophil counts were already significantly lower compared to controls. However, the alterations were not dose dependent. Therefore, all mentioned hematology changes were regarded as incidental and not treatment related.

Further detailed tables see attachment
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed. At the end of the administration period, in females of test group 3 (120 mg/kg bw/d) creatinine values were significantly decreased, but the values were within the historical control range (females, creatinine 26.9-35.4 µmol/L). In males of test group 2 (40 mg/kg bw/d) inorganic phosphate levels were significantly increased, but this alteration was not dose dependent. Therefore, both mentioned alterations were regarded as incidental and not treatment related.
In parental males of test groups 2 and 3 (40 and 120 mg/kg bw/d) T4 values were significantly increased. However, the means were within the historical control range (males, T4 44.65-73.22 mmol/L). Corresponding TSH values were not changed and within the historical control range (males, TSH 4.32-9.80 µg/L).

Further detailed tables see attachment
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation. Apart from transient salivation in three high-dose males and two mid-dose males, the open field observations did not reveal any test substance-related findings in male and female animals of all test groups. One high-dose male had an injury of hindlimbs during the open field observations, which was unrelated to treatment. There were no test substance-related findings in male and female animals of all test groups.
No test substance-related impaired parameters were observed in male and female animals of all test groups. The statistically significantly higher values of landing foot-splay test in males of test group 1 and 2 were considered as spontaneous in nature and not treatment-related, as there was no dose-response. In addition, the measured values were within the historical control range (HCD: 9.3 - 13.8 cm). Because of the hindlimbs injury of high-dose male No. 32 no measurement of grip strength of hindlimb and landing foot-splay test was performed with this animal.
No treatment-related, adverse change of motor activity (summation of all intervals) was observed in the male and female animals of all test substance-treated groups in comparison to the concurrent control values.
The mean number of beam interrupts of the high-dose parental males was statistically significantly above the concurrent control values during interval 1. However, the animals of the high-dose group habituated normally to the test conditions afterwards and all other groups including control were rather at the lower end of the historical control range for this interval (HCD: 989.6 – 1349.4). The high-dose value is within the historical control range, thus, considering the regular habituation, this slightly higher activity level is considered to be in the normal range of rat behavior.

Further detailed tables see attachment
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
When compared to control group 0 (=100%), the mean absolute weight of the liver was significantly increased in females of test group 3 (=119%). All other mean absolute weight parameters did not show significant differences when compared to the control group 0. When compared to control group 0 (=100%), the mean relative weights of the liver were significantly increased in males and females of test group 3. All other mean relative weight parameters did not show significant differences when compared to the control group 0.
The absolute liver weight increase in females (7.194 g) of test group 3 was within the historical control range (5.770 g – 7.568 g), as well as the significant increase in relative liver weight in females (3.144%) of test group 3 (historical control range 2.321 – 3.312%). The significantly increased relative liver weight in males of test group 3 (2.532%) was slightly above the historical control range (2.108% – 2.45%).
No histopathological correlate to the significantly increased relative liver weights were observed. Therefore, these findings were regarded as potentially treatment-related but not as adverse.

Further detailed tables see attachment
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were noted in the forestomach of male and female animals.
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.

Further detailed tables see attachment
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in the stomach of male and females of test groups 2 and 3.
Forestomach:
Diffuse squamous hyperplasia characterized by a thickening of the epithelial layer including the Margo plicatus and diffuse hyperkeratosis was noted. The lesion was correlated with the macroscopical findings of a thickened Margo plicatus and thickened wall. Additionally, erosions and ulcerations were present in multiple males and females of the test group 3. The epithelium surrounding the ulcerations and erosions displayed a pronounced squamous hyperplasia and hyperkeratosis. An inflammatory infiltrate, consisting of mainly neutrophils, lymphocytes and plasma cells was seen in the underlying tissue, as well as a submucosal edema. The squamous hyperplasia seen in one control male and one male/female each in test group 1 is regarded as incidental and not-treatment-related. In addition, the erosion/ulceration in one control male is a spontaneous background lesion.
Glandular stomach:
Multifocal edema and hyperemia of the lamina propria, often accompanied by an attenuation of the overlying epithelial layer was noted in male and female animals of test groups 2 and 3 and is assumed as consequence of a local irritating effect of the test substance. Additionally, ulcerations and / or erosions without a dose-dependency were found in the glandular stomach of males and females correlating with macroscopic findings. Since no dose-dependency could be observed and animals affected in test group 1 and 2 did not display the described test substance related histological lesions in the forestomach (diffuse squamous hyperplasia, submucosal edema and erosion/ulceration) and glandular stomach (edema/hyperemia in the lamina propria), this finding is regarded as a not treatment-related spontaneous background lesion.
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.

Further detailed tables see attachment
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
general systemic
Effect level:
12 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects in highest dose observed
Key result
Critical effects observed:
no

The results in tabular form are provided in the attached background material as pdf. Results for all organ weights (absolute and relative) are provided as separate pdf.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Chales River Laboratories, Research Models and Services, Germany GmbH/ Charles River Laboratories, France
- Age at study initiation: 11 weeks (male animals); 10 weeks (female animlas)
- Housing: During the pretreatment period of the study, the rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECHNIPLAST, Hohenpeißenberg, Germany.
During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany, with the following exceptions:
• 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 13 in Polycarbonate cages type III.
- Diet (e.g. ad libitum): ad libitum (ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Details on route of administration:
The test substance solutions in drinking water were prepared in intervals, which took into account the analytical results of the stability verification.
For the preparation of the administration solutions the test substance was weighed in a graduated flask depending on the dose group, topped up with drinking water and intensely mixed with a magnetic stirrer.
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Based on the analytical results it is concluded that the substance is stable in drinking water over a period of 7 days at ambient temperature. All determined concentrations were in the range of 90% - 110% of the nominal concentration.
Duration of treatment / exposure:
After the acclimatization period, the test substance was administered to the parental animals orally by gavage.
Frequency of treatment:
once daily at approximately the same time in the morning
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
Parental animals
Mortality
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

Clinical observations
A cageside examination was conducted at least once daily for any signs of morbidity,
pertinent behavioral changes and/or signs of overt toxicity. All animals were checked daily for any abnormal clinical signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal.

Food consumption
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions: Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals). Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20. Food consumption of the females which gave birth to a litter was determined for PNDs
1-4, 4-7, 7-10 and 10-13. 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.

Body weight data
In general, the body weight of the male and female parental animals was determined 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.
The following exceptions are notable for the female animals:
During the mating period the parental females were weighed on the day of positive evidence 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 4, 7, 10 and 13.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume;

Detailed clinical observations
Detailed clinical observations (DCO) were performed in all animals prior to the
administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined: 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.

Functional observational battery
A functional observational battery (FOB) was performed in the first five male and the first 5 female animals with litter per group (in order of delivery) at the end of the administration period starting at about 10.00 h. The FOB started in a randomized sequence with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

Home cage observations:
The animals were observed in their closed home cages (for a short period: about 10-30 seconds); during this period, any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: posture, tremors, convulsions, abnormal movements, gait, other findings

Open field observations:
The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined: behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/stereotypes, gait, activity/arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/color), rearing within 2 minutes, other findings

Sensory motor tests/ reflexes:
The animals were then removed from the open field and subjected to following sensory motor or reflex tests: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), other findings, grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test

Motor activity assessment
The Measurement of motor activity (MA) was measured at the end of the administration period in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group. Motor activity (MA) was measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For the duration of the measurement the animals were placed in new clean polycarbonate cages with a small amount of bedding. Eighteen beams were allocated per cage. The number of beam interrupts were counted over 12 intervals of 5 minutes per interval. The sequence in which the animals were placed into the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and was finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.

Clinical Pathology
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units. The parameters listed below were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.

Hematology
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, mean corpuscular volume, mean corpuscular Hemoglobin, mean corpuscular Hemoglobin concentration, Platelet count, Differential blood, Reticulocytes, Prothrombin time.

Clinical chemistry
Parameters: Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase), Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase), Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase), γ-Glutamyltransferase (GGT) (γ -glutamyl) peptide: aminoacid-γ- glutamyl-transferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, choloesterol, bile acids.

Thyroid Hormones
Blood samples were taken from all surplus pups or 2 preferably female pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling.
All generated serum samples were frozen at -80°C until measurement.
Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4).
T4 ELISA was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.
Sacrifice and pathology:
Parental animals

Necropsy
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

Organ weights
The following weights were determined in all animals sacrificed on schedule:
Anesthetized animals, Epididymides, Ovaries, Prostate, Seminal vesicles with coagulating glands, Testes, Thyroid glands (fixed) ,Uterus (with cervix).

The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus.

Organ/tissue fixation
Parental animals
The following organs or tissues of all parental animals were fixed in in 4% neutral-buffered formaldehyde solution or in modified Davidson`s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymides (modified Davidson`s solution), Esophagus, Extraorbital lacrimal glands, Eyes with optic nerve (modified Davidson`s solution), Femur with knee joint, Heart, Ileum, Jejunum (with Peyer`s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson`s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes (modified Davidson`s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus (uteri of all apparently nonpregnant animals or empty uterus horns were stained
according to Salewski E, 1964), Vagina.

Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.
All gross lesion, Adrenal glands, Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymides, Eyes with optic nerve, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Lymph nodes (axillary and mesenteric), Ovaries, Oviducts, Prostate gland, Peyer`s patches, Rectum, Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic, lumbar), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes, Thymus, Thyroid , glands, Trachea, Urinary bladder, Uterus, Vagina.
Statistics:
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Most animals of test group 3 as well as several animals of test group 2 (150 and 50 mg/kg bw/d) showed salivation after treatment (grade: slight to severe) at some occasions during the study period. In female animals the incidence was generally higher during gestation and lactation periods, affecting most test group 3 and several test group 2 females. These observations were considered to be treatment-related.
No further 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 groups. Spontaneous findings were seen in one high-dose male (No. 33) which showed red discharge in the right eye during mating days 2 - 4, 8 - 11 and postmating days 0 – 1 as well as one sperm positive mid-dose female (No. 121) which did not deliver F1 pups.

Further detailed tables see attachment
Mortality:
no mortality observed
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 female F0 generation parental animals in all test substance-treated groups showed no significant difference to the concurrent control during the entire study.
The statistically significantly lower body weight change in the mid-dose females during PND 7 - 10 was considered as spontaneous in nature and not as treatment-related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the F0 males in all dose groups (15, 50 and 150 mg/kg bw/d) as well as low and mid-dose females was not influenced by the treatment throughout the study.
Food consumption of the high-dose F0 females was statistically significantly below the concurrent control values during the entire premating period (up to 7%) while it remained unchanged during gestation and lactation periods.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males of test group 3 (150 mg/kg bw/d) prothrombin time (Hepatoquick’s test, HQT) was significantly reduced, but the mean was within the historical control range (males, HQT 37.4 - 40.9 sec). Therefore, this alteration was regarded as incidental and not treatment-related.

Further detailed tables see attachment
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males of test groups 1 and 2 (15 and 50 mg/kg bw/d) cholesterol values were significantly changed (test group 1 lower, test group 2 higher) compared to controls, but the alteration was not dose-dependent. Therefore, this alteration was regarded as incidental and not treatmentrelated.

Further detailed tables see attachment
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observational battery (FOB)
Home cage observations:
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
Open field observations:
Two male animals of dose group 3 (Nos. 33 and 34 - 150 mg/kg bw/d) showed slight (area around the mouth was moist) and severe (mouth very wet, wet paws) salivation, respectively.
All other male and female animals of all dose groups (15, 50 and 150 mg/kg bw/d) did not show any abnormalities.
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 higher value of the landing foot splay test in females of dose group 2 was considered as spontaneous in nature and not treatment related.
Motor activity measurement (MA)
No treatment-related changes of motor activity data was observed in all male and female animals of all dose groups (15, 50 and 150 mg/kg bw/d) in comparison to the concurrent control group. Overall activity levels and habituation to the test environment corresponded to the age of these animals, if usual biological variation inherent in the strain of rats used for this experiment was considered.
The isolated statistically significantly decreased numbers of beam interrupts in the males of dose groups 1 and 2 during interval 12 were not related to the dose and did not influence the overall activity levels and habituation. Thus, they were not considered to be related to the test substance.

Further detailed tables see attachment
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
None of the mean absolute or relative weight parameters showed significant differences when compared to the control group.

Further detailed tables see attachment
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were noted in the stomach and duodenum. 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.

Further detailed tables see attachment
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in forestomach and duodenum of male and female animals of test groups 2 and 3.
Duodenum: In the duodenum, a thickening of the mucosa characterized by an increase in villus height, was observed, which correlated to the macroscopic finding “dilation”. One female animal of test group 3 showed a focal hyperplasia of the duodenal mucosa.
Forestomach: Diffuse squamous hyperplasia (correlating with the macroscopic findings “margo plicatus, thickened”) and the presence of erosion/ulcer (correlating often with the macroscopic finding “focus”) were noted in the forestomach of male and female animals.
No treatment-related findings were noted in the glandular stomach and no correlate was found for the macroscopically observed discoloration in two test group 3 male animals. 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 morphology of the testicular interstitium and the stages of spermatogenesis in the testes of males of the high dose (150 ppm) were comparable to those of the controls.

Further detailed tables see attachment
Details on results:
Thyroid hormones:
In the F0 parental males of test groups 2 and 3 (50 and 150 mg/kg bw/d) T4 values were significantly higher compared to controls. However, the means were within the historical control range (males T4 44.87-88.29 nmol/L). Additionally, neither a change of thyroid weight nor any histopathological findings in the thyroids were noted. Therefore, the higher T4 values in parental males were regarded as incidental and not treatment-related.
In male and female pups at PND13 (test groups 11, 12 and 13; 15, 50 and 150 mg/kg bw/d), no treatment-related alterations of T4 levels were observed.

Further detailed tables see attachment

Key result
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.
Key result
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: irritation in forestomach and duodenum
Key result
Critical effects observed:
no

The results of clinical observation, body weights and organ weights, functional observation battery, oestrus cycle, haematology, clinical chemistry, gross pathology and histopathology are provided in tabular in the attached background material as pdf.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
30 May - 29 Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
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 or test system 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.

Further details / tables see attachments.
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 bw/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)

Further details / tables see attachments.
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
Description (incidence and severity):
Further details / tables see attachments.
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 bw/day group males on Week 13

Further details / tables see attachments.
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 mg/kg bw/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.

Further details / tables see attachments.
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)

Further details / tables see attachments.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Further details / tables see attachments.
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
Key result
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
Key result
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, behaviour effects, food consumption, estrus cycle and histopathology findings see "Attached background material".

 

Endpoint:
sub-chronic toxicity: oral
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
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to and including the highest dose group
Remarks on result:
other: CAS 2478-10-6, OECD TG 408
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
150 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Remarks on result:
other: CAS 2478-10-6, OECD TG 422
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
15 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: irritation in forestomach and duodenum
Remarks on result:
other: CAS 2478-10-6, OECD TG 422
Critical effects observed:
no
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:
OECD TG 408 (Read-across)
System:
gastrointestinal tract

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: inhalation
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 May 1974 to 07 May 1976
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Study was conducted prior to the advent of GLP regulations (1982).
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Principles of method if other than guideline:
Groups of 99-100 male and female rats were exposed 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
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-Hydroxyethyl acrylate
- Analytical purity: 94 %
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system 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: ambient
- Humidity: ambient
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
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 ppm
No. of animals per sex per dose:
99 - 100
Control animals:
yes, concurrent vehicle
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
Description (incidence and severity):
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.

Further details / tables see attachment.
Mortality:
no mortality observed
Description (incidence):
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.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weight data are presented in the table below, please see section "Any other information on results incl. tables". 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.

Further details / tables see attachment.
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:
effects observed, non-treatment-related
Description (incidence and severity):
Further details / tables see attachment. (gross pathology tables)
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- 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.

Further details / tables see attachment.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no significant differences between control and exposed groups.

Further details / tables see attachment.
Urinalysis findings:
no effects observed
Description (incidence and severity):
- 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.

Further details / tables see attachment.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- 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.

Further details / tables see attachment.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
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. (see Histopathological findings: non-neoplastic).

Further details / tables see attachment.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 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.

Further details / tables see attachment.

- 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.
 
- 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.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
A variety of neoplasms considered spontaneous in origin occurred in a number of tissues or organs of control and exposed groups of rats. As expected, mammary fibroadenomas and pituitary adenomas were the most frequently occurring neoplasms in female rats of all groups. Adrenal pheochromocytomas, pancreatic acinar adenomas, pituitary adenomas and subcutaneous fibromas were the most common neoplasms in the male rats of all groups.
Neoplasms, all of which were considered spontaneous in origin, occurred in the following organs and tissues: liver, lung, pancreas, kidney, testes, preputial gland, stomach, small and large intestine, spinal cord and peripheral nerves, pituitary gland, subcutaneous tissue, mammary tissue, integument, ear canal, oral cavity, adrenal gland, lymph nodes, spleen, brain, adipose tissue, thyroid, ovary, uterus, clitoral gland and vagina.
Statistical analyses revealed no exposure-related increases in the incidence of any of these neoplasms in HEA exposed rats compared to control rats. Statistical analyses also revealed no differences between the temporal or total incidence of HEA-exposed rats bearing benign neoplasms, malignant neoplasms, or all types of neoplasms combined compared to control rats.

Further details / tables see attachment.
Key result
Dose descriptor:
NOAEC
Remarks:
systemic effects
Effect level:
0.024 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: high dose
Dose descriptor:
NOAEC
Remarks:
local effects
Effect level:
0.002 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Key result
Critical effects observed:
no

All results in tabular form are provided in the attached background material as pdf.

Table 1a-b: Mean body weights of male and female rats exposed to vapors of 2 -Hydroxyethyl acrylate for up to 23 months (males) and 24 months (females)

 

1a: Days 0 -251

Days on Test

0

5

7

12

19

26

33

40

54

68

96

131

159

194

223

251

Males

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.0

ppm

299.8

(100)

314.4

(100)

322.2

(100)

343.4

(100)

375.3

(100)

393.3

(100)

416.1

(100)

437.6

(100)

466.9

(100)

488.0

(99)

522.6

(99)

548.3

(99)

569.0

(99)

594.6

(98)

557.3

(98)

608.2

(98)

5.0

ppm

*290.3

(99)

*308.7

(99)

320.4

(99)

*334.6

(99)

*364.1

(99)

392.5

(99)

*406.8

(99)

*422.4

(99)

*453.3

(99)

*473.4

(99)

*501.0

(99)

*528.0

(99)

*532.8

(99)

*546.4

(99)

555.6

(99)

*553.5

(99)

0.5

ppm

*287.0

(100)

*301.7

(100)

*309.3

(100)

*334.2

(100)

*357.0

(100)

379.7*

(100)

*391.9

(100)

*410.2

(100)

*437.6

(99)

*455.4

(99)

*495.3

(99)

*514.4

(97)

*519.9

(96)

*536.8

(95)

*541.5

(95)

*546.7

(94)

Females

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.0

ppm

224.1

(100)

221.0

(100)

222.7

(100)

232.8

(100)

241.8

(100)

247.3

(100)

259.3

(100)

266.7

(100)

277.6

(100)

287.2

(100)

302.6

(100)

293.7

(99)

309.3

(99)

331.2

(99)

337.0

(98)

336.9

(97)

5.0

ppm

*216.9

(100)

224.2

(100)

*230.6

(100)

235.0

(100)

243.7

(100)

*260.7

(100)

265.8

(100)

266.3

(100)

*287.0

(100)

*299.7

(100)

*312.1

(99)

*318.8

(99)

325.2

(99)

327.6

(98)

336.8

(98)

337.0

(98)

0.5

ppm

*219.2

(99)

222.2

(99)

220.0

(99)

230.3

(99)

241.7

(99)

*255.1

(98)

258.4

(98)

266.0

(98)

281.3

(98)

290.4

(98)

307.8

(98)

*320.7

(98)

309.5

(98)

327.4

(98)

332.2

(98)

336.4

(98)

1b: Days 286- 723

Days on Test

286

314

342

377

405

433

468

496

532

552

585

620

648

675

702

723

Males

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.0

ppm

632.7

(98)

642.8

(98)

648.9

(97)

654.3

(86)

658.0

(85)

661.4

(83)

664.1

(83)

628.3

(83)

593.0

(43)

604.0

(38)

577.2

(31)

587.1

(24)

558.3

(20)

521.8

(16)

518.8

(14)

 

5.0

ppm

*578.8

(99)

*583.4

(99)

*607.7

(98)

*615.2

(87)

*621.0

(86)

*621.0

(84)

*602.0

(78)

*543.4

(60)

589.2

(48)

589.3

(47)

599.0

(43)

587.0

(40)

557.7

(34)

507.2

(29)

535.3

(19)

 

0.5

ppm

*570.6

(93)

*575.5

(93)

*589.2

(93)

*596.0

(84)

637.8

(83)

*625.1

(84)

*627.7

(81)

611.4

(77)

587.3

(60)

573.9

(47)

558.7

(36)

559.4

(24)

520.5

(17)

549.1

(10)

518.9

(09)

 

Females

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.0

ppm

349.8

(97)

348.8

(95)

356.3

(96)

363.5

(86)

372.2

(86)

387.6

(85)

395.2

(85)

395.8

(85)

393.6

(73)

401.4

(67)

401.2

(61)

410.7

(52)

430.9

(48)

437.3

(38)

456.5

(29)

442.2

(20)

5.0

ppm

349.4

(95)

350.9

(95)

369.7

(93)

376.1

(83)

373.3

(83)

385.9

(82)

395.4

(81)

*359.7

(74)

387.6

(63)

395.8

(57)

417.7

(52)

413.3

(46)

421.9

(41)

404.3

(35)

406.9

(30)

411.0

(27)

0.5

ppm

351.9

(96)

351.6

(94)

351.6

(92)

367.2

(81)

*400.2

(81)

389.2

(78)

400.9

(76)

397.1

(73)

396.8

(73)

409.6

(63)

405.6

(55)

405.8

(47)

412.0

(41)

418.3

(31)

425.0

(23)

422.9

(20)

*: Significant difference from control data using analysis of variance and Dunnett's test p < 0.05.

( ) : Indicates numbers of rats weighed.

Day 0: Pre-exposure data

Table 2a-b: Body weights, organ weights measured and organ/body weight ratios for male (a) and female (b) rats exposed to vapors of 2 -Hydroxyethyl acrylate 5 days / week for 12 months

(a) males:

Exposure level (ppm)

Sex

Body weight g

Organ Weights (g and g/100 g Body Weight)

Brain

Heart

Liver

Kidneys

Testes

g

g/100g

g

g/100g

g

g/100g

g

g/100g

g

g/100g

0

M

633

1.94

0.31

1.60

0.25

17.83

2.82

4.40

0.70

4.35

0.69

M

584

2.02

0.35

1.54

0.26

12.76

2.18

3.48

0.60

4.36

0.75

M

670

1.97

0.29

1.68

0.25

17.46

2.61

4.60

0.69

4.18

0.62

M

635

1.96

0.31

1.73

0.27

15.89

2.50

4.12

0.65

3.88

0.61

M

583

1.95

0.33

1.66

0.28

11.75

2.02

3.13

0.54

2.55

0.44

Mean

± S.D.

621 ± 37

1.97 ± 0.03

0.32 ± 0.02

1.64 ± 0.07

0.26 ± 0.01

15.14 ± 2.75

2.42 ± 0.32

3.95 ± 0.62

0.63 ± 0.07

3.87 ± 0.76

0.62 ± 0.12

5.0

M

582

1.97

0.34

1.58

0.27

15.29

2.63

3.72

0.64

4.27

0.73

M

550

1.87

0.34

1.60

0.29

13.41

2.44

3.24

0.59

3.98

0.72

M

566

1.84

0.33

1.50

0.26

13.34

2.36

3.54

0.63

4.19

0.74

M

533

1.90

0.36

1.49

0.28

12.51

2.35

3.20

0.60

3.81

0.72

M

592

2.04

0.34

1.79

0.30

20.34

3.44

5.13

0.87

4.14

0.70

Mean

± S.D.

565*

±24

1.93 ± 0.08

0.34 ± 0.01

1.59 ± 0.12

0.28 ± 0.02

14.98 ± 3.17

2.64 ± 0.46

3.77 ± 0.79

0.66 ± 0.12

4.08 ± 0.18

0.72 ± 0.02

0.5

M

596

1.98

0.33

1.66

0.28

13.45

2.26

3.10

0.52

4.73

0.79

M

542

1.93

0.36

1.55

0.29

12.72

2.35

3.38

0.62

4.36

0.81

M

581

1.98

0.34

1.75

0.30

14.92

2.57

3.38

0.58

4.36

0.75

M

498

1.92

0.39

1.29

0.26

11.79

2.37

3.18

0.64

4.06

0.81

M

526

2.08

0.40

1.50

0.29

11.74

2.23

3.47

0.66

4.37

0.83

Mean

± S.D.

549*

±40

1.98 ± 0.06

0.36* ± 0.03

1.55 ± 0.17

0.28 ± 0.02

12.92 ± 1.32

2.35 ± 0.13

3.30 ± 0.15

0.60 ± 0.06

4.38 ± 0.24

0.80* ± 0.03

*: Statistically significant difference from control mean by analysis of variance and Dunnett's test p < 0.05.

(b) females:

Exposure level (ppm)

Sex

Body Body

weight g

Organ Weights (g and g/100 g Body Weight)

Brain

Heart

Liver

Kidneys

g

g/100g

g

g/100g

g

g/100g

g

g/100g

0

F

300

1.83

0.61

1.03

0.34

7.30

2.43

1.91

0.64

F

328

1.82

0.56

1.03

0.31

7.72

2.35

2.06

0.63

F

309

1.91

0.62

1.05

0.34

6.89

2.23

2.35

0.76

F

310

1.89

0.61

1.09

0.35

7.83

2.53

2.41

0.78

F

339

1.81

0.53

1.03

0.30

7.04

2.08

2.08

0.61

Mean

± S.D.

317 ± 16

1.85 ± 0.05

0.59 ± 0.04

1.05 ± 0.02

0.33 ± 0.02

7.36 ± 0.41

2.32 ± 0.18

2.16 ± 0.21

0.68 ± 0.08

5.0

F

359

1.83

0.51

1.17

0.33

7.50

2.09

2.47

0.69

F

332

1.83

0.55

0.12

0.34

7.69

2.32

2.43

0.73

F

337

1.69

0.50

1.04

0.31

7.98

2.37

2.27

0.67

F

376

1.85

0.49

1.22

0.32

8.90

2.37

2.53

0.67

F

307

1.83

0.60

1.09

0.36

7.29

2.38

1.87

0.61

Mean

± S.D.

342 ± 26

1.80 ± 0.07

0.53 ± 0.04

1.13 ± 0.07

0.33 ± 0.02

7.87 ± 0.63

2.30 ± 0.12

2.31 ± 0.27

0.68 ± 0.04

0.5

F

340

1.90

0.56

0.92

0.27

7.76

2.28

2.05

0.60

F

336

1.81

0.54

1.07

0.32

7.98

2.38

2.21

0.66

F

373

1.89

0.51

1.27

0.34

12.77

3.42

2.38

0.64

F

338

1.90

0.56

1.11

0.33

8.88

2.63

2.03

0.60

F

347

1.86

0.54

1.17

0.34

8.52

2.46

2.21

0.64

Mean

± S.D.

347 ± 15

1.87 ± 0.04

0.54 ± 0.02

1.11 ± 0.13

0.32 ± 0.03

9.18 ± 2.05

2.63 ± 0.46

2.17 ± 0.14

0.63 ± 0.02

The results of the presented toxicity study indicate that chronic inhalation exposure of rats to atmospheres containing 5 ppm HEA (corresponding to 0.024 mg/L) caused a minimal degree of toxicity; no toxicity was observed in rats exposed to 0.5 ppm HEA (corresponding to 0.0024 mg/L). There was no indication of a carcinogenic effect in the groups exposed to either 5 or 0.5 ppm HEA.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
24 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
Well performed and reported study.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Study with restrictions: no data on test substance purity. Study was conducted prior to the advent of GLP regulations (1982).
Principles of method if other than guideline:
Three groups of animals, each consisting of 25 male Sherman rats, were exposed to 5, 10, and 25 ppm of HEA vapours, respectively. The duration 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 two weeks and on the 25 ppm animals after one week of exposures.
GLP compliance:
no
Remarks:
(Study was conducted prior to the advent of GLP regulations)
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-Hydroxyethyl acrylate
- Analytical purity: no data
Species:
rat
Strain:
Sherman
Sex:
male
Details on test animals or test system and environmental conditions:
no details given
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: glass, dynamic exposure chambers of 160 liter volume
- Source and rate of air: Constant chamber airflow in each chamber was maintained by a rotary pump connected to the exhaust side of the chamber. The vapour was introduced into the air inlet located at the top of the chamber.
- Method of conditioning air: Vapour generation of HEA was achieved by aerosolizing the liquid with nitrogen, at calculated rates, in a temperature controlled vaporization flask. The aerosols were rapidly vaporized and then diluted with filtered room air to ambient temperature prior to its being drawn into the exposure chamber.
- Temperature, humidity, pressure in air chamber: ambient

TEST ATMOSPHERE
- Nominal concentration: The nominal concentration of HEA in a chamber was determined from the ratios of the rate of liquid compound being aerosolized (mg/minute) and the rate of total chamber airflow (L/minute; rate of nitrogen ejected from the aerosolizer plus that of the make-up air drawn from the room).
- Brief description of analytical method used: The analytical concentrations were obtained by absorbing the HEA with distilled water from known volumes of chamber atmospheres and analyzing the solution for HEA content with a gas-liquid chromatographic technique. The chamber concentrations were analyzed twice daily.
- Samples taken from breathing zone: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical concentrations were, in general, 20 to 40 % lower than the calculated concentrations being set up for the experiments indicating the recovery of HEA from the experimental atmospheres ranged from 60 to 80 % only. This poor recovery was partly due to the very low vapour pressure of HEA and, perhaps, its reactivity with the animal furs. Gas-liquid chromatographic analyses indicated that HEA vapours generated from the heated flask system at 80°C were identical to the liquid HEA in composition.
Mean analytical concentrations: 4.5 ± 1.1 ppm, 10.6 ± 1.4 ppm, and 22.5 ± 3.9 ppm
Duration of treatment / exposure:
20 exposures
Frequency of treatment:
7 hours/ day, 5 days/ week
Dose / conc.:
5 ppm (nominal)
Remarks:
corresponding to approx. 0.024 mg/L
Dose / conc.:
10 ppm (nominal)
Remarks:
corresponding to approx. 0.048 mg/L
Dose / conc.:
25 ppm (nominal)
Remarks:
corresponding to approx. 0.120 mg/L
No. of animals per sex per dose:
25 male rats per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure recovery period in satellite groups: 1 and 14 days post-exposure, respectively
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the exposures, the animals were observed closely for signs of irritation and toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: no data

BODY WEIGHT: Yes

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Interim sacrifices were performed on the 5 and 10 ppm groups after two weeks and on the 25 ppm animals after one week of exposures. Gross and microscopic examinations were carried out on all animals which succumbed during the experimental period. At termination of the exposure studies, all animals were sacrificed and examined likewise. The organ to body weight ratios for lung, liver, spleen, kidney and testes were ádetermined for the 5 and 10 ppm groups.
Statistics:
Statistical analyses were applied to all appropriate physical and biological data (t-test for significance).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- 5 ppm: During and after the exposures, the animals in the 5 ppm group did not show any adverse effects.
- 10 ppm: The animals in this group exhibited mild nasal irritation and discharge. After seven exposures, some animals also acquired lung rattles.
- 25 ppm: The responses of the animals to 25 ppm of HEA were eye and nasal irritation followed by dyspnoea and a bloated stomach (According to the authours these signs indicate that HEA is an upper respiratory tract irritant.). These conditions became more severe as the exposures prolonged. After one exposure, the animals began to lose body weight drastically and died probably of respiratory failure
Mortality:
mortality observed, treatment-related
Description (incidence):
- 5 ppm: No mortality observed
- 10 ppm: One animal died after 15 exposures
- 25 ppm: A total of eight animals died during the 10 exposure days and an addition of nine animals died after the termination of the exposures. Only three animals survived and recovered from the exposures.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 5 ppm: The growth and the body weight gain of the low-dose animals were similar to those observed in the control animals.
- 10 ppm: The mean body weights of the animals of the 10 ppm group showed, in general, a trend of loss during the five exposure days of the week, but a rapid recovery or a gain during the two no-exposure weekend days. Similar trends of loss and gain of mean body weights were observed repeatedly throughout the four week studies. It was noted that the mean terminal body weight of rats exposed to 10 ppm of HEA for twenty days was significantly lower than that of the control animals.
- 25 ppm: After one exposure, the animals began to lose body weight drastically.
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:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The organ to body weight ratios of liver and kidneys were significantly higher in exposed animals of the 10 ppm group. Similar elevation of liver to body weight ratio was also seen in the 5 ppm HEA groups. On the contrary, the heart to body weight ratio for the animals in the 5 ppm group was significantly lower.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See description "Histopahtological findings: non-neoplastic".
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Sacrificed animals:
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. With 14 days recovery, there was a lower incidence of corneal ulceration at the 10 ppm and 5 ppm level. One control rat had an ulcerative corneal lesion of unknown etiology.
Focal ulcerative rhinitis was present in 7 and 4 rats at 25 ppm and 10 ppm, respectively. This lesion was not present in the 14 days recovery rats on the above doses or in any 5 ppm and control rats.
Most HEA exposed rats and control rats had chronic inflammatory cells in the lamina propria of the trachea and larynx. In addition, 6, 4, and 6 rats at 25 ppm, 10 ppm, and 5 ppm, respectively, had superimposed acute reactions involving the mucosa of the trachea. Chronic-active tracheitis was not present in the control and was interpreted as resulting from HEA exposure. Chronic-active laryngitis was present in 7, 6, and 3 animals at 25 ppm, 10 ppm, and control levels, respectively. The lesion was not evident at the 5 ppm level.

- Deceased animals:
There were 17 spontaneous deaths in the group of 25 rats at the 25 ppm level, there was a high incidence of focal acute bronchopneumonia in these rats. Evaluation of the respiratory system was most difficult due to the high incidence of chronic murine pneumonia in the control and HEA exposed rats. Therefore, it was impossible to evaluate subtle pulmonary lesions with regard to HEA exposure.

The myocardial and testicular lesions observed were considered spontaneous and not related to HEA exposure.
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEC
Remarks:
systemic effects
Effect level:
ca. 0.12 mg/L air (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: high dose
Key result
Dose descriptor:
LOAEC
Remarks:
local effects
Effect level:
ca. 0.024 mg/L air (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no

Conclusions:

- HEA produced ulcerative keratitis at levels of 25 ppm, 10 ppm, and 5 ppm.

- Focal ulcerative rhinitis resulted from HEA exposure at 25 ppm and 10 ppm.

- 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.

- Chronic-active tracheitis in the HEA-exposed rats was interpreted as being compound-related at the 25 ppm, 10 ppm, and 5 ppm levels.

- Lesions at the 25 ppm level were more severe than those at the 10 ppm and 5 ppm levels.

- Bronchopneumonia and severe upper respiratory lesions were responsible for the spontaneous deaths at the 25 ppm HEA exposure level.

- Fourteen days recovery did not significantly reduce the number of lesions observed, except for the absence of ulcerative rhinitis.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
24 mg/m³
Study duration:
subacute
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

For the endpoint repeated dose toxicity via oral route a Weight of Evidence Approach (WoE) was conducted according Annex XI, Section 1.2 of the REACH regulation.

There is evidence from several independent sources available to perform the hazard assessment for this endpoint. The studies reported as Robust Endpoint summaries provide information on repeated dose toxicity from different species and routes of oral exposure (via diet or oral gavage).

For this WoE Approach studies performed with 2-hydroxyethyl acrylate (HEA, CAS 818-61-1) and the structurally analogue ester Hydroxypropyl acrylate (HPA, CAS 2918-23-2) have been investigated to receive a final conclusion on repeated-dose toxicity via the oral route.

There are two sub-chronic repeated dose oral toxicity studies available for HEA. In the first study, groups of male and female Sherman strain rats (10/sex/group) were maintained for 100 days on a diet containing 0, 0.03. 0.1 or 0.3 % HEA (equivalent to doses of 0, 20, 65, and 196 mg/kg body weight/day for males and 0, 30, 102, and 305 mg/kg body weight/day for females). Treatment did not cause any adverse effects as judged by general appearance and behaviour, growth, food consumption, serum, urea, nitrogen and alkaline phosphatase determinations, final average body and organ weights, organ/body weight ratios, and gross and microscopic examination of tissues. The NOAEL for male and female rats was approx. 196 – 305 mg/kg bw/day (Dow Chemical Co., 1967). This study meets most of the demands from the current OECD 408 guideline (2018) but lesser parameters have been investigated like only two clinical parameters and no haematology parameters for the test groups have been reported. No urinalysis was conducted. However, the study was well documented, pathological and histopathological examinations were performed and the exposure duration is comparable to the requests of the OECD TG 408 study.

In the second study, groups of male and female Beagle dogs (2/sex/group) were maintained for 97 days on a diet containing 0.06, 0.2, or 0.4 % HEA in diet (equivalent to doses of 21, 60 and 125 and 22, 63 and 131 mg/kg body weight/day for males and females respectively). There was no evidence of adverse effects as judged by general appearance and behavior, weight gain, food consumption, hematological values, examination of bone marrow, urea nitrogen content and alkaline phosphatase activity in the serum, serum bromsulfophthalein, serum glutamic oxalacetic transaminase and serum glutamic pyruvic transaminase tests, final average body and organ weights, and gross and microscopic examination of tissues. The NOAEL was approx. 125 -131 mg/kg bw/day (Dow Chemical Co., 1967). This study meets most of the demands from the current OECD TG 409 (1998) but the animal number is only half of those required in the current OECD TG 409, lesser haematological and clinical parameters have been investigated and no urinalysis was conducted. However, the study was well documented and the exposure duration is comparable to the requests of the OECD TG 409 study.

In a study according OECD 422, 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 (BASF SE, 2020). Control animals were dosed daily with the vehicle water. The duration of treatment covered a 5 weeks in-life period (males) including 14 days mating (mating pairs were from the same test group) as well as a 2-weeks premating period (females), 14 days mating period, 9 days post-mating period in one female (for no evidence of sperm), the entire gestation and about 3 weeks of lactation period in females up to one day prior to the day of scheduled sacrifice of the animals.

A detailed clinical observation (DCO) was performed in all animals before the start of the administration period and, as a rule, thereafter at weekly intervals. Water consumption of the F0 parents was determined. Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for GD 0 - 7, 7 - 14, 14 - 20 and PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13. Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on GD 0, 7, 14 and 20, on the day of parturition (PND 0) and on PND 1, 4, 7, 10 and 13. Estrous cycle data were evaluated for F0 generation females over a two 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 and 13. Their viability was recorded. At necropsy on PND 13, all pups were sacrificed with CO2 under isoflurane anesthesia (except the selected pups for blood sampling) and examined macroscopically for external and visceral findings. Anogenital distance measurements were conducted and all surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13. 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 animals per sex and group towards the end of the administration period. Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement. 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. 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.

Regarding clinical examination, many male and female animals of the mid-dose group and all male and female animals of the high-dose group (40 and 120 mg/kg bw/day) showed salivation immediately after dosing (up to 2 hours post dosing) during the treatment period. It is likely, that this temporary finding was induced by a local affection of the upper digestive tract. Although salivation is not an adverse finding per se, in the present study it most likely resulted from the considerable irritating potential of the test item which caused erosions/ulcerations in the stomach of a number of mid- and high-dose animals.

Otherwise the test item caused no clinical signs of toxicity. Particularly, in the in-depth investigations including the detailed clinical observation (DCO), the functional observational battery (FOB) and the measurement of motor activity (MA) no treatment-related differences to control were observed at any dose level.

Food consumption was generally comparable to the concurrent control in all treated groups, except for the high-dose female animals throughout lactation. Overall the high-dose females consumed about 14% less food than the control females during this study section.

Mean body weights were generally not influenced by the treatment. Body weight gain was lower in all treated groups at the beginning of exposure (though statistically significant only in the high-dose parental males during study days 0 – 7), suggesting that the animals needed a few days to get adapted to the bolus gavage of this irritating compound. After this adaptation the mean body weight change was generally comparable between concurrent control and treatment groups during the remaining study.

Concerning clinical pathology and thyroid hormone values, no treatment-related, adverse effects were observed up to a dose of the compound of 120 mg/kg bw/day.

Regarding pathology, the target organ was the stomach. Mild to moderate squamous hyperplasia and erosions and/or ulcerations were found in the forestomach of males of the mid-dose group (40 mg/kg bw/day) and animals of both sexes of the high-dose group (120 mg/kg bw/day), correlating with the thickened Margo plicatus, the thickened wall and the foci found in gross pathology. Additionally, a submucosal edema with inflammatory infiltrates was noted in animals of test group 3. In the glandular stomach a minimal to mild hyperemia/edema in the lamina propria often accompanied by an attenuation of the overlying epithelial layer was noted in animals of both sexes of test group 3. When the above described findings in the glandular stomach and the forestomach coincided, they were regarded as treatment-related and adverse local effects. In the glandular stomach of some males and females also erosions and ulcerations were noted, without showing a dose-dependency, but correlating with macroscopic findings. Since there was no dose response and animals affected in test group 1 and 2 did not display the above described related histological lesions in the forestomach and glandular stomach at large, this finding is regarded as a spontaneous background lesion and not treatment-related.

All other pathology 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. Furthermore, neither the synchrony of the morphology of the estrous cycle in ovaries, uterus, cervix, and vagina nor the stages of seminiferous tubules were altered.

Although the impact of the irritating potential of the test item was detected as local effects, one of the distinguished downstream consequences of the observed gastrointestinal pathology is pain, causing considerable distress and impairment of general condition in the affected animals. Collectively, salivation and gastrointestinal pathology indicate distinct parental toxicity in the high-dose animals and mild parental toxicity in the mid-dose animals.

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

Overall, pre-postnatal development was not influenced by the test item, as there were neither significant changes of pup survival or well-being nor pup body weight/body weight gain. However, there were two findings which require more in-depth consideration.

The statistically significantly shifted in sex ratio in the high-dose group on PND 0 (males 62.8*[*p<=0.05] versus [vs.] 43.8 in control and females 37.2* vs. 56.2 in control) was slightly outside the historical control range (HCD: 36.6% - 60.5% (males), 41.5% - 63.4% (females)). Nevertheless, it was assessed as not treatment-related, because pup necropsy at PND 13 revealed no morphological evidence for virilization and none of the other endocrine-sensitive parameters (e.g. pup weight, AGD, nipple retention) gave any indication of a test substance-related effect in any of the dose levels (see below). Thus, these values were most likely outliers and considered to be spontaneous in nature and not treatment related.

The apparently lower pup survival rate (83.6% vs. 99.1% in control, statistically non-significant) in the high-dose group was caused by the two individual dams 132 and 138. Dam 132 had a large litter (14 pups) and was not able to nurse all her pups properly. Consequentially 5 pups died from undernutrition within 3 days after they were born. Histopathology revealed a liver torsion in this animal, which presumably has essentially contributed to this insufficient nursing behavior. Dam 138 had only one pup, which also died from undernutrition 3 days after it was born, thus contributing to the lower pup survival percentage in a disproportionate fashion. This single pup death was most likely an incidental event. Thus, the pup losses for both dams were considered to be unrelated to treatment.

Neither determination of anogenital distance/index nor the count of nipple/areola anlagen revealed any treatment-related changes up to and including a dose level of the test item of 120 mg/kg bw/day.

In conclusion, the oral administration of HEA by gavage to male and female Wistar rats 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.

The structurally analogue HPA was evaluated in a Reproduction/Developmental Toxicity Screening Test according to OECD 422 and GLP (BASF 2018). The substance 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/day). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle 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. After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm 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 animals before the start of the administration period and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for GD 0-7, 7-14, 14-20 and PND 1-4, 4-7, 7-10 and 10-13. Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on GD 0, 7, 14 and 20, on the day of parturition (PND 0) and on PND 4, 7, 10 and 13. Estrous cycle data were evaluated for F0 generation females over a two week 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 and 13. Their viability was recorded. At necropsy on PND 13, all pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings. Anogenital distance measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen were counted. Blood samples were taken from all surplus pups or 2 preferably female pups per litter 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 animals per sex and group towards the end of the administration period. Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement. 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. 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. At the 150 mg/kg bw/day dose group the following observations were recorded: 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. In the mid dose group minimal thickening of the wall of the duodenum correlating to the macroscopically observed dilation in 2/10 male animals and minimal diffuse squamous hyperplasia of the forestomach in 6/10 male and female animals. No observations in the low dose-group were reported. In all dose groups no test substance-related adverse findings were reported for the F1 animals. The NOAEL for general, systemic toxicity of HPA was 150 mg/kg bw/day for male and female rats. Based on pathological findings in forestomach and duodenum in F0 parental rats of both sexes at 150 and 50 mg/kg as well as corresponding reductions of food consumption in F0 females at 50 mg/kg bw/day a NOAEL of 15 mg/kg bw/day was determined for local effects in the gastrointestinal tract. The NOAEL for fertility and reproductive performance was 150 mg/kg bw/day for the F0 parental rats. The NOAEL for developmental toxicity in the F1 progeny was 150 mg/kg bw/day.

In a study according to OECD TG 408 and GLP (Charles River Laboratory, 2018), HPA 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, thyroid hormones (T3, T4 or TSH levels), sperm morphology or differential counts. Test substance-related higher food consumption was noted in the 100 mg/kg bw/day group males and females (Days 1–90), and in the 30 mg/kg bw/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 bw/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 the 100 mg/kg bw/day group males. Higher mean kidney weights were present in the 100 mg/kg bw/day group males, and lower mean thymus weights were seen in the >10 mg/kg bw/day group females, both were considered not treatment-related.. 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 bw/day for a minimum of 90 days was well tolerated at all dosages. Therefore, the NOAEL was considered to be 100 mg/kg bw/day.

Conclusion:

It has been shown that HEA induces no test-item related systemic effects after administration via diet to Sherman rats or beagle dogs for 100 or 97 days, respectively. The obtained NOAEL in rats was 196-305 mg/kg bw/day after dietary administration, which were the highest doses tested in males and females, respectively. In these older studies via dietary administration the limit dose was not tested which might underestimate the hazard potential of HEA after oral exposure.

After administration of HEA via oral gavage to Wistar rats in a study according to OECD TG 422 the target organ identified was the GI tract and effects have been reported which are related to the irritating potential of the test item. Very similar results have been reported for the structurally analogue substance HPA in a study according OECD 422 after oral gavage to Wistar rats. Here, the GI tract was also the target organ due to the irritating nature of HPA. In both OECD TG 422 studies no effects have been observed on fertility and reproductive performance in the F0 generation and no adverse effects have been reported on development of the F1 generation up to postnatal day 13.

Results from the 90-day repeated oral dose study which was performed according OECD TG 408 with HPA revealed the absence of systemic effects in Wistar rats after oral gavage. A NOAEL of 100 mg/kg bw/day was derived, which was the highest dose tested. The dose selection for the OECD TG 408 study performed with HPA was based on the results of the 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 bw/day). The effects in the forestomach and duodenum were still observed in the mid dose (50 mg/kg bw/day), 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.

ECHA requested in their decision on compliance check on HEA (24thJuly 2019) to perform a repeated dose oral toxicity study according to OECD TG 408 in rats. Since new data from a study according OECD TG 422 for HEA are available which show very similar results compared to the OECD TG 422 study with the structurally analogue substance HPA after oral gavage in Wistar rats, read-across to the OECD TG 408 study performed with HPA is considered suitable to fulfil the REACH requirements for HEA for the endpoint repeated dose oral toxicity. In both studies according to OECD TG 422 the NOEALs for parental toxicity were comparable (i.e. 12 and 15 mg/kg bw/day for HEA and HPA, respectively). HEA and HPA caused similar adverse effects in the gastro-intestinal tract which was the only target organ. The observed effects can be related to the irritating potential of both substances. No adverse effects have been reported for fertility and reproductive performance for the F0 generation. Therefore, read-across from the available OECD TG 408 for HPA is considered suitable to fulfil the REACH requirements for this endpoint. Further testing for the sub-chronic exposure of HEA is not indicated, also because of animal welfare reasons.

 

Repeated dose toxicity: Inhalation route

In the key chronic inhalation study (Dow Chemical Co., 1979), 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) of test substance 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. 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.

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. The duration 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.

The test result from the sub-acute study is supported by data from a structural analogue:

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.

Repeated dose toxicity: Dermal route

There are no experimental data available on subchronic and chronic toxicity of 2-hydroxyethyl acrylate by the dermal route of exposure. But due to animal welfare reasons and since the substance is corrosive and a skin sensitizer, no long-term testing by the dermal route is planned.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available 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 fourteenth time in Regulation (EU) No 2020/217.