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REACH

2-ethylhexyl acrylate

EC number: 203-080-7 | CAS number: 103-11-7

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

2-Ethylhexyl acrylate Extended One-Generation Reproduction Toxicity Study in Wistar Rats Administration via the Diet without extension to produce the F2 generation

2-Ethylhexyl acrylate was administered to groups of 25 male and 25 female healthy young Wistar rats (F0 parental generation) as a homogeneous addition to the food in different concentrations (0, 1500, 5000 and 12500 ppm). These concentrations were reduced to 50% (750, 2500 and 6250 ppm) during lactation. This dietary adjustment, derived from historical body weight and food consumption data, maintained the dams at the desired target doses of 2-Ethylhexyl acrylate during this period of increased food intake.

Stability, correct concentrations and homogeneous distribution of 2-Ethylhexyl acrylate in the diet were all confirmed through analytics.

For the male animals the overall mean dose of 2-Ethylhexyl acrylate throughout all study phases and across all cohorts was approx. 119 mg/kg body weight/day (mg/kg bw/d) in the 1500 ppm group, approx. 357 mg/kg bw/d in the 5000 ppm group and approx. 998 mg/kg bw/d in the 12500 ppm group. For the female animals the overall mean dose of 2-Ethylhexyl acrylate throughout all study phases and across all cohorts was approx. 135 mg/kg bw/d in the 1500 ppm group, approx. 453 mg/kg bw/d in the 5000 ppm group and approx. 1136 mg/kg bw/d in the 12500 ppm group.

There were no test substance-related mortalities or adverse clinical observations noted in any of the treatment groups in the F0 parental animals and F1 offspring. In particular, regularly conducted detailed clinical observations revealed no effects.

The high concentration of the test substance (12500 ppm) produced signs of adverse local effects which were subsequently conveyed to some subsequent systemic effects in the F0 parental rats and F1 adolescents.

In the 12500 ppm and partly also 5000 ppm F0 parental animals food consumption was reduced during several episodes of the study. No comparable findings were noted in the F1A and F1B offspring. Although they might be related to the irritating properties of the test item (see pathology), those changes were minor and followed a patchy pattern, thus they did not qualify as adverse test substance-related effects by themselves.

Body weights and body weight change of the 12500 ppm male F0 and F1 rats were consistently and, in many parts of the study significantly, below the concurrent control across all cohorts and study periods, including terminal body weight. Female F0 and F1 rats were less sensitive against this effect, significant body weight decreases were limited to single episodes during the study, overall the pattern and severity of changes pointed toward a borderline outcome.

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the test compound of 12500 ppm in F0 as well as F1A rats of both sexes.

Regarding pathology, target organs were the glandular stomach and the kidneys.

Thus, under the conditions of the present extended one-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 5000 ppm (about 357 mg/kg bw/d in males, 453 mg/kg bw/d in females) in the F0 parental and the F1 adolescent/adult rats, based on evidence for local toxicity in the gastrointestinal tract which conveyed to systemic toxicity such as decreased body weight/body weight gain across generations and cohorts, at the LOAEL (Lowest Observed Adverse Effect Level) of 12500 ppm (about 998 mg/kg bw/d in males, 1136 mg/kg bw/d in females).

Study on the Inhalation Toxicity of 2-Ethylhexyl Acrylate as a Vapor in Rats (3-Months Study).

In a valid 90-day inhalation study (BASF, 1989) Wistar rats were administered in a whole-body exposition on 6 hours per day, 5 days per week, to 2-EHA vapour at concentrations of 0 ppm, 10 ppm, 30 ppm or 100 ppm (approximately 0.075 mg/l, 0.225 mg/l or 0.750 mg/l for the treatment groups) (2-EHA purity 99.7%). The study design was conducted according to OECD 413 (1981). Compared to the actual version of the test guideline, validity is restricted in that food consumption was not recorded, lung tissues were not perfused, and laryngopharynx was not examined. Histopathologic examination was carried out on 31 organs/tissues of high dose and control animals. The lungs, nasal cavity, thyroid and parathyroid glands, trachea and liver from all animals/all test groups were subjected to histopathology examination.

There were no treatment-related premature deaths. During exposure period animals of the high and mid dose groups exhibited lethargy and ptosis. Body weight gain was lower in both sexes of the high dose during and at the end of the study. A transiently reduced body weight gain was observed in mid dose females. From day 21 onwards mean body weight (absolute) was lower in high dose males compared to the control group. This parameter was not significantly altered in any other group at any time point during the study. Activities of ALAT and alkaline phosphatase were elevated in high dose females. In high dose males and females lower levels of total protein, albumin and glucose were demonstrated. Reduced protein and albumin values were also seen in each sex of the mid dose groups.

Absolute liver weight was reduced in high dose males and relative adrenal weights were lower in high dose males and females compared to the control groups. The microscopic examination revealed no lesion other than a focal or diffuse degeneration of the olfactory epithelium of the cranial nasal cavity in animals of both sexes of the high and mid dose groups. All rats of the 100 ppm group showed degeneration of the olfactory mucosa in the anterior part of the nasal cavity. The incidence of degeneration of the olfactory mucosa but not the severity was increased in mid dose rats. No treatment-related lesion of the nasal cavity was diagnosed at the low dose level.

Degeneration of the olfactory epithelium was characterised by a reduction of cell layers, reduction or loss of apical cytoplasmic structures such as olfactory knobs and microvilli, and by necrosis. Identification of the remaining olfactory mucosa cells was not possible. Occasional mitosis were present.

In detail, degeneration of the olfactory mucosa was diagnosed in the anterior part of the nasal turbinates (level 1) in all high dose rats and in four males and four females of the mid dose group. At the high dose level, the degeneration affected the olfactory mucosa diffusely in the dorsal and dorsolateral area, and the severity was mainly moderate. Mid dose animals showed small areas of degeneration of the dorsolateral olfactory mucosa of minimal severity. At level 2 of the turbinates, degeneration was diagnosed in all high dose rats, one mid dose male, two female and one female of the low dose group, and in one control group male. In the high dose group, the degeneration was diffuse in the dorsal and dorsolateral region, whereas in the other groups the degeneration was focal. The severity was minimal to marked in the high dose group, and mainly minimal in the other groups. Slight degeneration of the olfactory mucosa of the level 3 was diagnosed in one male and one female each of the high dose group. 2 -EHA induce no lesions of the trachea and the lungs.

The No-observed-adverse-effect-level (NOAEL) for the local effects on the respiratory tract was 10 ppm (0.075 mg/l), and the NOAEC for systemic effects was 30 ppm (0.226 mg/l).

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 all close all

Reference 1

Endpoint:: sub-chronic toxicity: oral
Study period:: 19 Mar 2021 to 2023
Data waiving:: other justification
Justification for data waiving:: other:
Justification for type of information:: Due to unscheduled delays in the contract laboratory, the final report for this study is not yet available, so only the draft report is available so far.
Qualifier:: according to guideline
Guideline:: other: OECD Guideline 443
Version / remarks:: 25 Jun 2018
GLP compliance:: yes
Specific details on test material used for the study:: 2-Ethylhexyl acrylate purity 99.8 %
Species:: rat
Strain:: Wistar
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 34 days
- Housing: 5 animal (Polysulfonate cages Typ 2000P (H-Temp):During premating, male animals after mating (post-mating), females after weaning and animals of cohorts 1A and 1B); 1 animal (Polycarbonate cages type III During mating, gestation, lactation) Exceptions: During mating: 1 male/1 female per cage; During lactation, rearing up to weaning: 1 dam with her litter
- Diet (e.g. ad libitum): Mouse and rat maintenance diet “GLP”, Granovit AG, Kaiseraugst, Switzerland; ad libitum
- Water (e.g. ad libitum): drinking water, ad libitum
- Acclimation period: 7 days before administration, during the acclimatization period, the animals will become accustomed to the environmental conditions and to the diet.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:: oral: feed
Details on oral exposure:: Due to the weaknesses in the OECD422 based on technical difficulties, especially the preparation of the test substance in the application media (concentration analysis showed large variabilities and dosing formulations did not meet the acceptability criteria), the study is of limited reliability. To avoid these difficulties in requested OECD 443 including range-finding study it was decided to perform those tests by dietary route. Since 2EHA is an irritant monomer the local inflammatory effects in the stomach might also be reduced by application of the test substance via the diet.

PREPARATION OF DOSING SOLUTIONS:
Preparation frequency:
The test substance preparations is prepared at intervals which guarantee that the test substance concentrations in the diet will remain stable.
Storage conditions of the preparations:
The test-substance preparations are split in aliquots and stored at -18°C in order to offer new test-substance preparations. The food in the food hoppers are changed at least every three days (after the preparations have reached room temperature)
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Analytical verifications of the stability of the test substance in the diet for a period of 32 days in freezer following 4 days at room temperature was verified before the start of the study.
Analyses were carried out 4 times during the study. At the beginning, in the middle and toward the end of the study, as well as for female diets once in the lactation period (Concentration control (mid dose) and Homogeneity analyses (low + high dose)).
Duration of treatment / exposure:: The F0 animals, with the exception of the controls, were fed diets supplying the appropriate amounts of the test substance for approximately 10 weeks prior to breeding and continuing through breeding (up to two weeks), and for a maximum of 4 post-mating weeks (males) or gestation (three weeks) and lactation (three weeks) for females. Selected F1 offspring (cohorts 1A and 1B) were maintained on the test diet until one day before sacrifice.
Frequency of treatment:: daily
Dose / conc.:: 1 500 ppm
Remarks:: overall mean dose: male: 119 mg/kg bw/d + female 135 mg/kg bw/d
Dose / conc.:: 5 000 ppm
Remarks:: overall mean dose: male: 357mg/kg bw/d + female 453 mg/kg bw/d
Dose / conc.:: 12 500 ppm
Remarks:: overall mean dose: male: 998 mg/kg bw/d + female 1136 mg/kg bw/d
No. of animals per sex per dose:: 25
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale:
In a modified 421 study (reproduction/developmental toxicity screening test, project No. 81R0903/00S059), 2-Ethylhexyl acrylate was administered to each 10 male and female Wistar rats at concentrations in the diet of 0, 3000, 7500 and 15000 ppm. During lactation, concentrations in the diet of the F0 females were reduced to 50%. In the highest test group (15000 ppm), mean food consumption and body weight development were the most affected parameters.
Food consumption was (statistically significantly) reduced in males (up to 20.2% below control) and females (up to 14.5% below control) of test group 3 and in males of test group 2 (up to 18% below control) during premating. During gestation, females of test group 3 recovered to values comparable to control whereas during lactation, mean food consumption showed a higher variation.
Body weight (change) was decreased in males of test group 3 with a body weight loss in the beginning of the study (body weight change during study days 0-7: -2 g versus 12.6 g in control). However, these males recovered and gained weight towards the end of the study (4.8% below control). For females of test group 3, body weight (change) was not affected during premating but showed a statistically significant decrease during gestation (8.4% below control). During lactation, body weight of these animals recovered almost to control values.
In this extended one-generation study, pretreatment/premating will be prolonged to 10 weeks and animals are younger compared to the 421-study leading to a higher substance intake in the beginning of the study. In this study, the highest concentration in the diet aims to reach limit dose considering all the different study phases.
Based on the above-mentioned results and at the request of the sponsor, the following concentrations in the diet were selected:
1500* ppm as low concentration
5000* ppm as mid concentration
12500* ppm as high concentration
*During the lactation period the 2-Ethylhexyl acrylate concentrations in the diet of the F0 was reduced to 50%. This dietary adjustment derived from historical body weight and food consumption data will maintain the dams at the desired target doses of 2-Ethylhexyl acrylate during this period of increased food intake.

- Other:
F0 generation parental animals and F1 pups:
Male and female animals, aged about 4 weeks when supplied, will be used as F0 generation parental animals. After an acclimatization period of at least 7 days, these animals will be tended to for at least 10 weeks.
Then the F0 animals will be mated. The female F0 animals will be allowed to deliver and rear their pups (F1 generation pups) until postnatal days (PND) 4 or 21. The male F0 generation parental animals will be sacrificed during rearing (see 2.5. time schedule). The female F0 generation parental animals will be sacrificed after weaning of the F1 generation pups. All F0 females will be sacrificed after weaning.
Observations and examinations performed and frequency:: MORTALITY:
A check for moribund and dead animals was made twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays.

CAGE SIDE OBSERVATIONS: (for F0 generation parental animals and selected F1 rearing animals)
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. If such signs occur, the animals will be examined several times daily. Abnormalities and changes will be documented for each animal.
The parturition and lactation behavior of the dams was generally evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver or umbilical cord not cut) will be documented on an individual dam basis.
On weekdays (except Saturdays, Sundays and public holidays) the parturition behavior of the dams will be inspected in the afternoons in addition to the evaluations in the mornings.
The day of parturition is considered to be the 24-hour period from about 15:00 h of one day until about 15:00 h of the following day. Departures from this procedure may occur on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS:
All F0 parental animals and F1 animals in cohorts 1A and 1B were subjected to detailed clinical observations (DCO) outside their cages once before the beginning of the administration period (day 0 only for F0 parental animals) and subsequently once per week (in the morning). For observation, the animals will therefore be removed from their cages and placed in a standard arena (50 × 37.5 × 25 cm).
The scope of examinations and the scoring of the findings observed will be based on the current index of findings in ToxLIMS software and includes but is not limited to the following parameters listed:
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

BODY WEIGHT:
In general, the body weight of the male and female F0 parental animals and F1 rearing animals was determined once a week at the same time of the day (in the morning), if possible.
The following exceptions are notable for the female parental animals:
During the mating period of the F0 parental animals, the 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 after parturition (PND 1) and on 4, 7, 10, 14, 18 and 21.
Females without litter and after weaning (PND 21) were weighed once a week

FOOD CONSUMPTION
Generally, food consumption was determined once a week (over a period of 7 days) for male and female F0 parental animals and F1 rearing animals, with the following exceptions:
•Food consumption was not determined after the 10th premating week (male F0 and F1B animals) and during the mating period (male and female F0 parental animals).
• Food consumption of the F0 females with evidence of sperm was determined weekly for GD 6-7, 13-14 and 19-20.
• Food consumption of the F0 females, which gave birth to a litter was determined for PND 3-4, 6-7, 9-10, 13-14, 17-18 and 20-21.
• Food consumption of females showing no positive evidence of sperm in the vaginal smear was determined once a week during this mating interval as were the males
• Food consumption of females without litter and after weaning (PND 21) was determined once a week

WATER CONSUMPTION AND COMPOUND INTAKE:
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

DETAILED CLINICAL OBSERVATION DCO)
Detailed clinical observations were performed in all F0 parental animals once before the administration and supsequently once per week and in cohorts 1A and 1B 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:
1. Abnormal behavior in handling
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos (Protruding eyeball)
15. Assessment of the feces excreted during the examination (appearance/consistency)
16. Assessment of the urine excreted during the examination
17. Pupil size

CLINICAL PATHOLOGY:
Clinical Pathology in F0 parental and cohort 1A animals
Samples were withdrawn from the first 10 surviving F0 parental (females with litter, corresponding males) and the first 10 surviving cohort 1A males and females per group at termination. Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood examinations were carried out in a randomized sequence. The list of randomization instructions was compiled with a computer.
In the afternoon preceding the day of urinalysis, the animals will be individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens will be examined in a randomized sequence (the list of randomization instructions will be compiled with a computer).

The following parameters were examined:

Hematology:
Leukocytes, Erythrocytes, Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelets, Differential blood count, Reticulocytes,
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.
Prothrombin time (Hepato Quick’s test) (HQT)

Clinical chemistry:
Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Serum g-glutamyl transferase, Sodium, Potassium, Chloride, Inorg. phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins, Triglycerides, Cholesterol

Hormone evaluations:
T4 (thyroxine), THS

Splenic lymphocyte subpopulation analysis:
Ten males and females per group of cohort 1A were used to perform a splenic lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells) using one half of the spleen, the other half of the spleen being preserved for histopathological evaluation.

Urinalysis:
Volume, Color, Turbidity, pH value, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Microscopy of sediment
Sacrifice and pathology:: Necropsy
All F0 parental animals and all cohort 1A 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.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights:
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (final body weight), Adrenal glands (fixed), Brain, Caudae epididymides, Epididymides, Heart, Kidneys, Liver, Lymph nodes, axillary (10 animals per sex per group, cohort 1A animals only), Lymph nodes, mesenteric (10 animals per sex per group, cohort 1A animals only), Ovaries, Pituitary gland (fixed), Prostate (ventral and dorsolateral part together, fixed), Testes, Seminal vesicles including coagulating glands (fixed), Spleen, Thymus (fixed), Thyroid glands (with parathyroid glands) (fixed), Uterus with cervix.
All paired organs will be weighed together (left and right).

Organ/Tissue fixation:
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Adrenal glands, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymis, left (modified Davidson’s solution), Esophagus, Eyes with optic nerve (modified Davidson’s solution), Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Liver, Lungs, Lymph nodes, axillary, Lymph nodes, mesenteric, Mammary gland (male and female), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Pituitary gland, Prostate, Rectum, Sciatic nerve, Seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), Spleen, Stomach (forestomach and glandular stomach), Target organs, Testis, left (fixed in modified Davidson ´s solution), Thymus, Thyroid glands (with parathyroid glands), Trachea, Urinary bladder, Uterus, Vagina, Vas deferens.

The left testis and left epididymis of all male F0 parental and Cohort 1A animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters analysis .
In case of macroscopic findings in the right testis or right epididymis, this testis as well as the corresponding epididymis were fixed for histopathological examination and the left testis and epididymis were used for sperm analysis.

The uteri of all cohabited female F0 parental animals were examined for the presence and number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method). Then the uteri were rinsed carefully in physiologic salt solution (0.9 % NaCl). When the examinations were completed, the uteri were transferred to the Pathology Laboratory for further processing.
Spleens of 10 animals per sex per group of cohort 1A were split in two comparable parts (transversally). One part of the spleen was fixed in 4% neutral buffered formaldehyde and afterwards embedded in paraplast. The other part of the spleen was frozen at -80o C, being used to perform a splenic lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells).

Histopathology:
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below:
All gross lesions, Adrenal glands, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymis, left (modified Davidson’s solution), Esophagus, Eyes with optic nerve (modified Davidson’s solution), Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Liver, Lungs, Lymph nodes, axillary, Lymph nodes, mesenteric, Mammary gland (male and female), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyriod glands, Pituitary gland, Prostate, Rectum, Sciatic nerve, Seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), Spleen, Stomach (forestomach and glandular stomach), Target organs, Testis, left (fixed in modified Davidson ´s solution), Thymus, Thyroid glands (with parathyroid glands), Trachea, Urinary bladder, Uterus, Vagina, Vas deferens.

The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004).
A correlation between gross lesions and histopathological findings was attempted.
Special attention was given to stages of spermatogenesis in the male gonads.
Special attention was also given to the synchrony of the morphology in ovaries, uterus, cervix, and vagina to the estrous cycle status.
An immunohistochemial stain for a2μ in the kidneys was performed exemplarily in 2 control and 2 high dose male animals of F0 animals (animal no. 2, 13, 77, 92).
Reproductive organs of all F0 animals suspected of reduced fertility were subjected to histopathological investigation.
A differential ovarian follicle count (DOFC) was conducted in test groups 10 and 13 (Cohort 1A females) according to Plowchalk et.al. (1993).

Differential Ovarian Follicle Count (DOFC) in cohort 1A females
A differential ovarian follicle count (DOFC) was conducted in test groups 10 and 13 (cohort 1A females) according to Plowchalk et.al. (1993) by the Test Facility. In general, sections were prepared with 2 - 3 μm thickness and serial sections were taken every 100 μm to complete about 20 cut levels across the whole ovarian tissue. For the counting of primordial and growing follicles, HE-stained slides were prepared from all cut levels. Counting was performed on slides digitalized with a Hamamatsu NanoZoomer 2.0 slide scanner using the Hamamatsu viewing software (NDP.view).
Statistics:: DUNNETT test (two-sided): Food consumption (parental and rearing animals), body weight and body weight change (parental DUN NETT test (two-sided) animals, rearing animals and pups); for the pup
weights, the litter means were used), gestation days, duration of sexual maturation (days to vaginal
opening, days to preputial separation), anogenital distance, anoqenital index
FISHER'S EXACT test (one-sided): Male and female mating indices, male and female FISHER'S EXACT test (one-sided) fertility indices, gestation index, females mated, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups
WILCOXON test (one-sided+) with BONFERRONHOLM: Mating days until day O pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development
WILCOXON test (one-sided-) with BONFERRONHOLM: Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, lactation index
WILCOXON test (two-sided): % live male day x, %live female day x
KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided): Number of cycles and Cycle Length
KRUSKAL-WALLIS and WILCOXON test: Clinical pathology parameters
KRUSKAL-WALLIS and WILCOXON test: Weight of the anesthetized animals and absolute and relative organ weights
WI LCOXON test ( one-sided -): DOFC
KRUSKAL-WALLIS and WILCOXON test: Weight of the anesthetized animals and absolute and relative organ weights
KRUSKAL-WALLIS-H and WILCOXON: Weight of the anesthetized animals and absolute and relative organ weights
Clinical signs:: effects observed, non-treatment-related
Description (incidence and severity):: Clinical observations for males and females:
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the groups.

Clinical observations for females during gestation of F1 litters:
There were no test substance-related clinical findings noted in any female of all dose groups during the gestation period for F1 litter.
One low-dose female (No. 139) had blood in bedding on GD 22.

Clinical observations for females and offspring during lactation of F1 litters:
There were no test substance-related clinical findings noted in all F0 females of all dose groups during the lactation period.
One high-dose female (189) had a complete litter loss and in one mid-dose female (169) all pups were stillborn.

Detailed clinical observations:
Male and female animals of all dose groups (1500, 5000 and 12500 ppm) did not show any abnormalities.
Mortality:: no mortality observed
Description (incidence):: There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: Mean body weights of low-dose F0 males were comparable to the concurrent control values throughout the study. Mean body weight of mid-dose F0 male animals was significantly decreased on study day 105 by -6%. In male animals of the high dose the mean body weights were significantly below the concurrent control values from study day 21 onwards with a maximum by -9% on study day 91.

Mean body weights of the low-and mid-dose F0 females were comparable to the concurrent control values throughout the entire study. In F0 females of the high dose the mean body weight was significantly decreased on GD 20 by -6%, while the mean body weight during premating and lactation were comparable to the concurrent control group.

In comparison to concurrent control, statistically significant decreases and increases of body weight change were observed in all treated F0 male animals as specified below:

• Low-dose males: decrease on study days 42-49 and 98-105, increase study days 91- 98 and 105-112
• Mid dose males: decrease on study days 0-7, 42-49, 77-84, 98-105, increase on study days 70-77, 91-98 and 105-112
• High-dose males: decrease on study days 0-7, 14-21, 28-35, 42-49, 49-56 and 98-105, increase on study days 91-98

Overall, body weight change from study day 0 to 112 was below concurrent control in all treatment groups, however, the decrease gained statistical significance only in the mid-dose males (-7%) and in the high-dose males (-11%).

Body weight change in females of low- and mid-dose group as well as body weight change during premating and lactation in females of high dose group was comparable to the concurrent control group during the entire study.

Body weight change in females of the high-dose group was significantly below to the concurrent control group on study days GD 0-7 (-17%), GD 14-20 (-16%) and GD 0-20 (-14%) during the gestation period.
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: Food consumption of the high-dose F0 males was statistically significantly below the concurrent control values on study days 14, 49, 56, 63 and 105 (maximum by -15% on study day 49) as well as of the mid-dose males on study days 49 (-13%) and 56 (-11%).

Food consumption of the low-, mid- and high-dose F0 females during premating period was comparable to the concurrent control.

Food consumption of the high-dose F0 females was statistically significantly below the concurrent control on GD 7 (-11%) and on GD 20 (-10%) as well as on PND 4 (-16%), PND 10 (-13%) and on PND 18 (-13%).

Food consumption of the low- and mid-dose F0 females during gestation and lactation period was comparable to the concurrent control values.
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 F0 females of test group 03 (12500 ppm) absolute large unstained cell (LUC) counts were significantly higher compared to controls. The values were within the historical control range (F0 females, LUC 0.00-0.02 Giga/L). Therefore, this change was regarded as incidental and not treatment related.
Clinical biochemistry findings:: effects observed, non-treatment-related
Description (incidence and severity):: No treatment-related, adverse changes among clinical chemistry parameters were observed.
At the end of the administration period, in F0 males of test group 03 (12500 ppm) alanine aminotransferase (ALT) activities were significantly decreased. However, this decrease was below 50% and therefore, this change was most probably due to a liver enzyme induction (PSD Guidance Document, 2007). This was regarded as treatment related but adaptive and not adverse.
In F0 females of test group 03 sodium levels were significantly lower compared to controls, but the values were within the historical control range (F0 females, sodium, 139.7-143.0 mmol/L).
Endocrine findings:: no effects observed
Description (incidence and severity):: Thyroid hormones
In F0 males and females no treatment related effect on the T4 and TSH values could be observed.

In F0 males T4 values in test groups 01, 02 and 03 (1500, 5000 and 12500 ppm) were significantly higher compared to controls. The change was not dose dependent, and all mean values were within the historical control range (F0 males, T4 40.44-72.39 nmol/L). TSH values in males of test groups 01, 02 and 03 were lower compared to study controls, but not statistically significantly. Again, all TSH values were within the historical control range (F0, TSH 3.49-10.31 μg/L). TSH mean value in the study controls was at the upper border of the historical control range. Therefore, the alterations of T4 and TSH in F0 males of test groups 01, 02 and 03 were regarded as incidental and not treatment related.
In F0 dams of test group 03 (12500 ppm) T4 values were significantly higher compared to controls, but the values were within the historical control range. The same was true for the TSH values among these individuals (F0 dams, T4 24.28-46.41 nmol/L; TSH 3.23-6.30 μg/L). Therefore, the T4 change in females of test group 03 was regarded as incidental and not treatment related.
Urinalysis findings:: effects observed, non-treatment-related
Description (incidence and severity):: No treatment-related, adverse changes among urinalysis parameters were observed.
At the end of the administration period, in F0 males of test group 03 (12500 ppm) the incidence of transitional epithelial cells and that of granulated and epithelial casts were significantly in-
creased. These findings were observed only in males, and they were most probably due to an α2u-Globulinuria which was regarded as a species-specific finding without human relevance (Hard, 2018)
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: The terminal body weight of males in test group 3 (12500 ppm) was significantly decreased (380.0g, -8.3%).

Absolute organ weights:
In males of test group 03 (12500 ppm) the absolute weight of heart (1.034g; -7.1%) and seminal vesicles (1.229g, -11.4%) was significantly decreased. The relative organ weights were not significantly decreased and therefore the changes in absolute weight of heart and seminal vesicles were regarded as secondary to the decreased terminal body weight.

Relative organ weights:
The significant increases of relative kidneys (0.631%, +6.8%; 0.654% +10.7%), brain (0.539%, + 5%; 0.552% +7.5%) and liver (2.267%; +6.5%; 2.383%; +12.0%) weights in males of test groups 02 (5000 ppm) and 03 (12500 ppm) were within the historical control range (kidneys: 0.577%-0.68%, brain:
0.591% - 0.61%, liver: 2.155% - 2.49%) and regarded as secondary to the decreased body weight.

The increase in absolute liver weight of females of test group 03 (12500 ppm) (6.614g, +7.8%) was slightly above the historical control range (5.600g - 6.530g), whereas the significant increase in relative liver weights (2.794%, +9.9%) in females is within the historical control range (2.47% - 2.833%).

No histopathological correlate could be detected. The finding was regarded as potentially treatment related but not adverse. All other mean relative weight parameters did not show significant differences when compared to the control group 00.
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: In 8 out of 25 female animals of test group 03 (12500 ppm) foci were detected in the glandular
stomach. This finding could be correlated to an increased incidence of erosion/ulcer detected in histopathology and was regarded as treatment related.
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.

Fertility:
The female animals (Nos. 103, 105, 108, 112, 134, 138, 148, 149, 172, 182, 184 and 197) which were not pregnant as well as their male mating partners (Nos. 3, 5, 8, 12, 34, 38, 48, 49, 72, 82, 84 and 97), did not show relevant gross lesions.
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: Treatment-related findings were observed in the glandular stomach of females and the kidneys of males.
The increased incidence of erosion/ulcer in the glandular stomach females of test group 03 (12500 ppm) correlated to the macroscopically detected foci in the glandular stomach and was regarded as treatment related.
In the kidneys of males of test group 03 (12500 ppm) a minimal to slight increase in basophilic tubules was detected. In most animals the kidneys were affected unilaterally. The increase in basophilic tubules was regarded as potentially treatment related but not adverse. An immunohistochemial stain for a2μ in the kidneys was performed with a negative result exemplarily in 2 control and 2 high dose male animals of F0 animals (animal no. 2, 13, 77, 92).
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.

Fertility:
The female animals (Nos. 103, 105, 108, 112, 134, 138, 148, 149, 172, 182, 184 and 197) which were not pregnant as well as their male mating partners (Nos. 3, 5, 8, 12, 34, 38, 48, 49, 72, 82, 84 and 97), did not show relevant histopathological findings consistent with impaired fertility.
: Key result
Dose descriptor:: NOAEL
Effect level:: 5 000 ppm
Based on:: test mat.
Sex:: male/female
Basis for effect level:: body weight and weight gain; other: local toxicity in the gastrointestinal tract
Remarks on result:: other: 5000 ppm: about 357 mg/kg bw/d in males, 453 mg/kg bw/d in females
Conclusions:: Thus, under the conditions of the present extended one-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 5000 ppm (about 357 mg/kg bw/d in males, 453 mg/kg bw/d in females) in the F0 parental and the F1 adolescent/adult rats, based on evidence for local toxicity in the gastrointestinal tract which conveyed to systemic toxicity such as decreased body weight/body weight gain across generations and cohorts, at the LOAEL (Lowest Observed Adverse Effect Level) of 12500 ppm (about 998 mg/kg bw/d in males, 1136 mg/kg bw/d in females).

Reference 2

Endpoint:: short-term repeated dose toxicity: oral
Type of information:: experimental study
Adequacy of study:: weight of evidence
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: comparable to guideline study
Remarks:: OECD 421 like
Guideline:: other: modified 421 study (repoduction/developmental toxicity screening test)
Principles of method if other than guideline:: Range-finding study for OECD 443 (EOGRTS)
GLP compliance:: no
Species:: rat
Strain:: Wistar
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Charles River Laboratories, Research Models ans Services, Sulzfeld, Germany
- Age at study initiation: 14-15 weeks (male); 13 weeks (female)
- Housing: Type of cage: Polysulfonate cages type 2000P (H-Temp)
Exceptions: During mating (males and females) and females during gestation, lactation and after weaning: Polycarbonate cages type III
No. of animals per cage: Polysulfonate cages: -During pretreatment: up to 5 animals per sex and cage; -During premating: 2 animals per sex and cage
Polycarbonate cages: 1 animal
Exceptions: -During mating: 1 male/1 female per cage; -During rearing up to PND 13: 1 dam with her litter
- Diet (e.g. ad libitum): Mouse and rat maintenance diet "GLP" Granovit AG, Kaieraugust, Switzerland
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:: oral: feed
Details on route of administration:: Due to the weaknesses in the OECD422 based on technical difficulties, especially the preparation of the test substance in the application media (concentration analysis showed large variabilities and dosing formulations did not meet the acceptability criteria), the study is of limited reliability. To avoid these difficulties in requested OECD 443 including range-finding study it was decided to perform those tests by dietary route. Since 2EHA is an irritant monomer the local inflammatory effects in the stomach might also be reduced by application of the test substance via the diet.
Details on oral exposure:: DIET PREPARATION:
The test substance preparations will be prepared at intervals which guarantee that the test substance concentrations in the diet wil remain stable.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Concerning analytics, sampling for homogeneity and concentration control analysis was performed at the start of the administration period and once during gestation as well as lactation. Samples of all concentrations were measured by the Analytical Laboratory of the test facility.
Duration of treatment / exposure:: 27 days (male); 62 days (female)
Frequency of treatment:: daily
Dose / conc.:: 3 000 ppm
Dose / conc.:: 7 500 ppm
Dose / conc.:: 15 000 ppm
No. of animals per sex per dose:: 10
Control animals:: yes, concurrent vehicle
Details on study design:: During lactation, concentrations in the diet of the F0 females were reduced to 50%. At the request of the sponsor and based on the results of a previous 28-day range-finding study, the dose levels were selected.
Observations and examinations performed and frequency:: OBSERVATIONS:
The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances.
A check for moribund and dead animals will be made twice daily from Mondays to Fridays and once daila on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS:
A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption of the F0 parents was determined regularly once weekly before and after the mating period, as well as in dams during gestation days 7, 14 and 20 and lactation days 4, 7, 10 and 13.

BODY WEIGHT:
In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0, 4, 7, 10 and 13.CAGE SIDE
Sacrifice and pathology:: GROSS NECROPSY:
All F0 parental were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, adrenal glands, epididymides, kidneys, liver, ovaries, prostate, seminal vesicles, spleen, testes and uterus with cervix.
Histological examination was performed on Adrenal glands, Kidneys, Liver, Spleen, Stomach (forestomach and glandular stomach) and macroscopic findings in both sexes.
Statistics:: Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used) , gestation days: DUNNETT (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 (one-sided)
Mating days until day O pc, % postimplantation loss, with stillborn, %perinatal lass: WI LCOXON ( one-sided+) BONFERRONI-HOLM
Implantation sites, pups delivered, pups liveborn, life pups day x, with viability Index, survival index: WI LCOXON ( one-sided-) BONFERRONI-HOLM
% live male day x, % live female day x: WI LCOXON (two-sided)
Number of cycles and cycle length: KRUSKAL-WALLIS (twosided) and WILCOXON (two-sided)
Clinical signs:: effects observed, non-treatment-related
Description (incidence and severity):: Test group 3 (15000 ppm):
No test substance-related, adverse clinical signs
One incidental finding assessed as not related to treatment: One F0 female animal (No. 136) showed a mass palpable through the skin (>3 cm) in the region of the throat during gestation (from GD 25 onwards) and was sacrificed in a moribund condition for animal welfare reasons during lactation (PND 8). The same animal had only one implant and a dead pup leading to a complete litter loss.
Test group 2 (7500 ppm):
One incidental finding assessed as not related to treatment: One female animal (No. 126) with an injury in the neck.
Mortality:: no mortality observed
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: Test group 3 (15000 ppm):
Decrease in mean body weight (change) (BW SD 28: 5% below control) in males with a body weight loss during SD 0-7 (-2 g versus 13 g in control).
Decrease in mean body weight in females during gestation (8% below control).
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: Food consumption was (statistically significantly) reduced in males (up to 20% below control) and females (up to 15% below control) of test group 3 and in males of test group 2 (up to 18% below control) during premating. During gestation, females of test group 3 recovered to values comparable to control whereas during lactation, mean food consumption showed a higher variation. The decrease in food consumption was assessed to be secondary due to the smell and taste of the test substance in the diet.
Gross pathological findings:: effects observed, non-treatment-related
Description (incidence and severity):: All gross lesions occurred individually and were considered incidental or spontaneous in nature and not related to treatment. One female animal of test group 3 (No. 136), sacrificed in a moribund state, showed macroscopically a mass of 50 mm in diameter in the cervical region correlating with a malignant myoepithelioma, and an enlarged spleen correlating with a massive extramedullary hematopoiesis. In addition, histopathology revealed in the liver a slight extramedullary hematopoiesis. These findings were most likely associated to the tumor and explain the moribund state of the animal; however, they were not considered treatment related.
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: Pathology:
Concerning pathology, in males of test group 3 (15000 ppm), a statistically significant relative weight increase of the kidneys (+10% above the control group) correlated with minimally to moderately increased eosinophilic droplets in the tubular epithelium in 8 out of 10 males. Increased eosinophilic droplets most likely represents an accumulation of α2u-globulin synthesized in the male rat liver, filtered by the glomerulus, and reabsorbed in the S2 segment of the proximal tubules. It is considered a treatment-related and male rat specific finding but does not represent a risk for humans since they do not synthesize this protein (Durham and Swenberg, 2013). Minimal tubular degeneration/regeneration in 2 out of 10 males and granular casts in 1 out of 10 were assessed as not relevant due to the low incidence and grading. Therefore, the findings in the male kidneys were considered treatment-related but not adverse. A statistically significant liver weight increase in males of test group 3 (+9% above the control group) was marginally above the historical control values and occurred without a histopathological correlate. Thus, it was regarded as treatment-related but not adverse. All other histopathological findings occurred individually and were considered incidental or spontaneous in nature and not related to treatment.
The mean absolute weight of the prostate in males of test group 3 (0.993 g) was statistically significantly decreased (-18%) compared to the control. This decrease was marginally below the historical control range (1.001 – 1.267 g), whereas the mean relative weight (0.276%) was within the normal range (0.264 – 0.33%) without a statistical significance and so it was regarded as not treatment-related. All other absolute and relative weight parameters did not show significant differences when compared to the control group 0.
Histopathological findings: neoplastic:: effects observed, non-treatment-related
Description (incidence and severity):: spontanous finding: myoepithelioma of the parotid glands in animal No. 136
: Key result
Dose descriptor:: NOEL
Effect level:: 7 500 ppm
Based on:: test mat.
Sex:: male/female
Basis for effect level:: body weight and weight gain; histopathology: non-neoplastic
Critical effects observed:: no

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 357 mg/kg bw/day
Study duration:: subchronic
Experimental exposure time per week (hours/week):: 168
Species:: rat
Quality of whole database:: Reliability 1, OECD TG study, GLP
System:: gastrointestinal tract

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Apr 1985 - 19 July 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

1981

Deviations:

yes

Remarks:

food consumption was not recorded, lung tissues were not perfused, and laryngopharynx was not examined.

GLP compliance:

yes

Limit test:

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. K. Thomae GmbH, D-7950 Biberach, Germany
- Age at study initiation: approximately 8-week old
- Weight at study initiation (mean): male rats: 223 g (213 - 234 g); female rats: 158 g (150 - 164 g)
- Housing: single
- Diet (ad libitum): Kliba Labordiaet Ratte/Maus A 343 10 mm Pellet, Klingentalmuehle AG, CH-4303 Kaiseraugst
- Water (ad libitum): tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12 h dark / 12 h light

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation chambers (glass/steel inhalation chambers) with a capacity of approx. 1.3 m3
- Method of holding animals in test chamber: animals in wire cages
- Temperature, humidity in air chamber: 22.8-23.6 °C, 36-41 %

TEST ATMOSPHERE
- Brief description of analytical method used: The test substance concentrations were checked intermittently (10 and 30 ppm) or continuously (100 ppm) by total hydrocarbons analyzers (THCA)/GC/FID.
- Samples taken from breathing zone: yes

EXPOSURE SYSTEM
The test and control animals were exposed to the test substance vapors and control air in a whole-body exposure system, respectively. All air streams, supply air and exhaust air of all test groups were adjusted by air flow meters (rotameters), measured continuously and recorded approximately every 2 hours. The air temperatures in the exposure systems were measured continuously with multipurpose thermometers (Diehl GmbH & Co) and recorded approximately every 2 hours. The pressures in the exposure systems were measured continuously (slanting tube pressure gage) and recorded once a day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Measured test substance concentrations were within ± 5% of the nominal concentrations (see table below).

Duration of treatment / exposure:

90 days

Frequency of treatment:

6 h/day; 5 days/week

Dose / conc.:

10 ppm

Remarks:

0.075 mg/L

Dose / conc.:

30 ppm

Remarks:

0.225 mg/L

Dose / conc.:

100 ppm

Remarks:

0.753 mg/L

No. of animals per sex per dose:

Groups of 10 male and 10 female rats per test concentration were exposed to test substance vapors in a whole-body exposure system on 6 hours per day, 5 days per week at 10, 30, and 100 ppm (corresponding to approximately 0.075 mg/L, 0.226 mg/L or 0.753 mg/L).

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale:
In order to obtain preliminary information about the profile of action of 2-EHA vapor, the maximum concentration during the 90-day study was to be within the saturation range of the test substance (estimated by extrapolation of the vapor-pressure curve: T = 20°C; p= 0.13 mbar = 130 ppm). For technical reasons (e.g. condensation phenomena and hence poor reproducibility with slight temperature fluctuations), the maximum concentration which could be reliably maintained under the study conditions was chosen to be 100 ppm. The minimum concentration of 10 ppm was based on the applicable MAC value for methyl acrylate (1984). The intermediate concentration was established between these two concentrations as 30 ppm in order to obtain any concentration-response relationships.

- Post-exposure period: none

- Control: An air control with 10 male and 10 female rats was run in parallel.

Positive control:

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Mortality and clinical signs were checked each day.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight determinations were conducted once a week.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of the animals in the control group and the high dose group were checked with a focusable hand-held slit lamp for changes in the refracting media at the beginning of the pre-flow period (day -8) and at the end of the study (day 83).
- Dose groups that were examined: high-dose, control

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the exposure period
- Anaesthetic used for blood collection: No
- How many animals: 10
- Parameters were examined:
The following hematological examinations were carried out on 10 animals per test group and sex at the end of the exposure period: Hemoglobin, erythrocytes, hematocrit, mean hemoglobin content per erythrocyte, mean cell volume, mean corpuscular hemoglobin concentration, platelets, leukocytes.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the exposure period
- How many animals:
- Parameters were examined:
The following clinicochemical examinations were carried out on 10 animals per test group and sex at the end of the exposure period: Total bilirubin, creatinine, urea, sodium, potassium, total protein, glucose, inorganic phosphate, calcium, chloride, triglycerides, cholesterol, albumine, globulins.
The following enzyme activities were determined: - Glutamic-pyruvic transaminase - Alkaline phosphatase - Glutamic-oxalacetic transaminase. Clotting analyses were performed: Prothrombin timne (Hepato Quick's test).

URINALYSIS: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

The animals were sacrificed at the end of the 3-month inhalation period and each animal was completely necropsied. Histopathologic examination was carried out on 31 organs/tissues of high dose and control animals. The lungs, nasal cavity, thyroid and parathyroid glands, trachea and liver from all animals/all test groups were subjected to histopathology examination.

Statistics:

T-test according to Williams.

WILLIAMS DA (1971 ). A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics 27: 103 - 117
WILLIAMS DA (1972 ). The comparisdn of several dose levels with a zero dose control. Biometrics 28: 519 - 531

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- 100 ppm (0.753 mg/L): Mild lethargy, eyelid closure during exposure.
- 30 ppm (0.225 mg/L): Mild lethargy and eyelid closure during exposure.
- 10 ppm (0.075 mg/L): As compared to control animals, no toxic effects which could be interpreted as being related to the test substance.

Mortality:

no mortality observed

Description (incidence):

There were no deaths during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 100 ppm (0.753 mg/L): Retarded body weight gain in both sexes on determination of the body weight change; retarded body weight gain in male animals only on determination of body weight.
- 30 ppm (0.225 mg/L): Slight retardation in body weight gain of the female animals.
- 10 ppm (0.075 mg/L): As compared to control animals, no toxic effects which could be interpreted as being related to the test substance.

Food consumption and compound intake (if feeding study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- 100 ppm (0.753 mg/L): Increase in glutamic-pyruvic transaminase activity and alkaline phosphatase activity of female rats; reduction of total proteins in female animals and very slightly in male rats; slightly reduced albumin concentrations in both sexes (not statistically significant); reduction of glucose level in female animals.
- 30 ppm (0.226 mg/L): Reduced total protein and albumin levels in both sexes; only a tendency to a reduction in albumin levels in female animals.
- 10 ppm (0.075 mg/L): As compared to control animals, no toxic effects which could be interpreted as being related to the test substance.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- 100 ppm (0.753 mg/L): Reduction in body weight resulted in darker coloration of liver parenchyma with indistinct lobular marking (decrease in liver lipids).
- 30 ppm (0.226 mg/L): No findings.
- 10 ppm (0.075 mg/L): No findings.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Nasal cavity:
Degeneration of olfactory mucosa was diagnosed. This degeneration was characterized by a reduction of cellular layers, reduction or loss of apical cytoplasmic structures such as olfactory knobs and microvilli, and by necrosis. Identification of the remaining olfactory mucosa cells was not possible. Occasional mitoses were present.
Level I: Degeneration of olfactory mucosa was diagnosed in all rats of 100 ppm group and in 4 males and 4 females each of 30 ppm group. In 100 ppm group, the degeneration affected the olfactory mucosa diffusely in the dorsal and dorsolateral area, and the severity was mainly moderate. In 30 ppm group, the degeneration affected only small areas of the dorsolateral olfactory mucosa and was minimal in severity.
LeveL II: Degeneration of olfactory mucosa was diagnosed in all rats of 100 ppm group, 1 male of group 2, 2 males and 1 femaLe of 10 ppm group 1, and 1 male of control group. In 100 ppm group, the degeneration was diffuse (dorsal, dorsolateral), whereas in the other groups, the degeneration was focal. The severity was minimal to marked in 100 ppm group, and mainly minimal in the other groups.
LeveL III: Degeneration of oLfactory mucosa was only diagnosed in one male and one female each of the 100 ppmgroup. The severity was slight.
Level IV: No degeneration of the olfactory mucosa was noted.

Liver
Fatty change, characterized by micro- to macrovesicular hepatocellular fat deposits was diagnosed in rats of all groups including controls.
The fatty change was mainly localized in zones 1 (periportal zone) and 2 (intermediate zone). In 5 males of 30 ppm group and in one male of 100 ppm group, fatty change was noted also in zone 3 (zone surrounding terminal hepatic venule). Generally, the severity of fatty change was higher in males than in females. In females, the severity of fatty change was similar in all groups. In males, the severity of fatty change was less in 100 ppmgroup when compared with the rats of the other groups.

Dose descriptor:

NOAEC

Remarks:

nasal irritation

Effect level:

0.075 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEC

Remarks:

systemic effects

Effect level:

0.226 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

Critical effects observed:

yes

Lowest effective dose / conc.:

0.225 mg/L air (analytical)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Concentrations in the inhalation chambers:

A study mean with standard deviation was calculated from the daily means of the individual chamber concentrations of the test groups:
		Concentration
     Nominal		       Measured
 [ppm]	[mg/L]		[ppm]    	[mg/L]
----------------------------------------------
  10	0.075		9.9 ± 0.47	0.0745
  30    0.226		30 ± 1.7	0.226
 100    0.753		100 ± 4.6	0.753
----------------------------------------------

Executive summary:

In a valid 90-day inhalation study (BASF, 1989) Wistar rats were administered in a whole-body exposition on 6 hours per day, 5 days per week, to 2-EHA vapour at concentrations of 0 ppm, 10 ppm, 30 ppm or 100 ppm (approximately 0.075 mg/l, 0.226 mg/l or 0.753 mg/l for the treatment groups) (2-EHA purity 99.7%). The study design was conducted according to OECD 413 (1981). Compared to the actual version of the test guideline, validity is restricted in that food consumption was not recorded, lung tissues were not perfused, and laryngopharynx was not examined. Histopathologic examination was carried out on 31 organs/tissues of high dose and control animals. The lungs, nasal cavity, thyroid and parathyroid glands, trachea and liver from all animals/all test groups were subjected to histopathology examination.

In detail, degeneration of the olfactory mucosa was diagnosed in the anterior part of the nasal turbinates (level 1) in all high dose rats and in four males and four females of the mid dose group. At the high dose level, the degeneration affected the olfactory mucosa diffusely in the dorsal and dorsolateral area, and the severity was mainly moderate. Mid dose animals showed small areas of degeneration of the dorsolateral olfactory mucosa of minimal severity. At level 2 of the turbinates, degeneration was diagnosed in all high dose rats, one mid dose male, two female and one female of the low dose group, and in one control group male. In the high dose group, the degeneration was diffuse in the dorsal and dorsolateral region, whereas in the other groups the degeneration was focal. The severity was minimal to marked in the high dose group, and mainly minimal in the other groups. Slight degeneration of the olfactory mucosa of the level 3 was only diagnosed in one male and one female each of the high dose group. 2-EHA induced no lesions of the trachea and the lungs, data of the pharynx/larynx were not available.

Table 2-EHA induced olfactory degeneration in rats from a 90-day inhalation study (BASF, 1989)

	Dose group	Control		10 ppm		30 ppm		100 ppm
	Sex	M	F	M	F	M	F	M	F
	No. of animals	10	10	10	10	10	10	10	10
Nasal cavity	Level 1 (anterior)					4	4	10	10
	Mean severity grade					1	1	2.9	3.1
	Level 2	1		2	1	1		10	10
	Mean severity grade	1		1	1	2		2.9	2.5
	Level 3							1	1
	Mean severity grade							2	2

Grading used 1 = minimal, 2 = slight, 3 = moderate, 4 = marked; M = male, F = female

Treatment-related microscopic lesions outside the respiratory tract were seen in the liver. Fatty change (at low and medium severity grades a common finding in well fed rats) occurred in rats of all dose groups and control groups. In high dose males, the severity of fatty change was less compared to other groups and the control. The mean severity grades of lipid accumulation in the periportal zone of the liver lobus decreased from 2.6 in control males to 1.0 in the high concentration males, a minimal change was also seen in high concentration females (1.0 versus 1.6 in controls). No indication of a peroxisomal proliferation was evident in electron microscopy. No treatment-related effects were evident at the low dose group.

The No-observed-adverse-effect-level (NOAEL) for local effects on the respiratory tract was 10 ppm (and 0.075 mg/l), and the NOAEC for systemic toxic effects was 30 ppm (and 0.226 mg/l).

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEC
: 226 mg/m³
Study duration:: subchronic
Experimental exposure time per week (hours/week):: 30
Species:: rat
System:: hepatobiliary
Organ:: liver

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Apr 1985 - 19 July 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

1981

Deviations:

yes

Remarks:

food consumption was not recorded, lung tissues were not perfused, and laryngopharynx was not examined.

GLP compliance:

yes

Limit test:

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Measured test substance concentrations were within ± 5% of the nominal concentrations (see table below).

Duration of treatment / exposure:

90 days

Frequency of treatment:

6 h/day; 5 days/week

Dose / conc.:

10 ppm

Remarks:

0.075 mg/L

Dose / conc.:

30 ppm

Remarks:

0.225 mg/L

Dose / conc.:

100 ppm

Remarks:

0.753 mg/L

No. of animals per sex per dose:

Control animals:

yes, sham-exposed

Details on study design:

Positive control:

Observations and examinations performed and frequency:

Sacrifice and pathology:

Statistics:

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mortality:

no mortality observed

Description (incidence):

There were no deaths during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Food consumption and compound intake (if feeding study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Dose descriptor:

NOAEC

Remarks:

nasal irritation

Effect level:

0.075 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEC

Remarks:

systemic effects

Effect level:

0.226 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

Critical effects observed:

yes

Lowest effective dose / conc.:

0.225 mg/L air (analytical)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Concentrations in the inhalation chambers:

A study mean with standard deviation was calculated from the daily means of the individual chamber concentrations of the test groups:
		Concentration
     Nominal		       Measured
 [ppm]	[mg/L]		[ppm]    	[mg/L]
----------------------------------------------
  10	0.075		9.9 ± 0.47	0.0745
  30    0.226		30 ± 1.7	0.226
 100    0.753		100 ± 4.6	0.753
----------------------------------------------

Executive summary:

In a valid 90-day inhalation study (BASF, 1989) Wistar rats were administered in a whole-body exposition on 6 hours per day, 5 days per week, to 2-EHA vapour at concentrations of 0 ppm, 10 ppm, 30 ppm or 100 ppm (approximately 0.075 mg/l, 0.226 mg/l or 0.753 mg/l for the treatment groups) (2-EHA purity 99.7%). The study design was conducted according to OECD 413 (1981). Compared to the actual version of the test guideline, validity is restricted in that food consumption was not recorded, lung tissues were not perfused, and laryngopharynx was not examined. Histopathologic examination was carried out on 31 organs/tissues of high dose and control animals. The lungs, nasal cavity, thyroid and parathyroid glands, trachea and liver from all animals/all test groups were subjected to histopathology examination.

In detail, degeneration of the olfactory mucosa was diagnosed in the anterior part of the nasal turbinates (level 1) in all high dose rats and in four males and four females of the mid dose group. At the high dose level, the degeneration affected the olfactory mucosa diffusely in the dorsal and dorsolateral area, and the severity was mainly moderate. Mid dose animals showed small areas of degeneration of the dorsolateral olfactory mucosa of minimal severity. At level 2 of the turbinates, degeneration was diagnosed in all high dose rats, one mid dose male, two female and one female of the low dose group, and in one control group male. In the high dose group, the degeneration was diffuse in the dorsal and dorsolateral region, whereas in the other groups the degeneration was focal. The severity was minimal to marked in the high dose group, and mainly minimal in the other groups. Slight degeneration of the olfactory mucosa of the level 3 was only diagnosed in one male and one female each of the high dose group. 2-EHA induced no lesions of the trachea and the lungs, data of the pharynx/larynx were not available.

Table 2-EHA induced olfactory degeneration in rats from a 90-day inhalation study (BASF, 1989)

	Dose group	Control		10 ppm		30 ppm		100 ppm
	Sex	M	F	M	F	M	F	M	F
	No. of animals	10	10	10	10	10	10	10	10
Nasal cavity	Level 1 (anterior)					4	4	10	10
	Mean severity grade					1	1	2.9	3.1
	Level 2	1		2	1	1		10	10
	Mean severity grade	1		1	1	2		2.9	2.5
	Level 3							1	1
	Mean severity grade							2	2

Grading used 1 = minimal, 2 = slight, 3 = moderate, 4 = marked; M = male, F = female

The No-observed-adverse-effect-level (NOAEL) for local effects on the respiratory tract was 10 ppm (and 0.075 mg/l), and the NOAEC for systemic toxic effects was 30 ppm (and 0.226 mg/l).

Endpoint conclusion

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

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Nov 1984 - 19 Feb 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report

Principles of method if other than guideline:

Since the epicutaneous application of 86.5 and 21 % 2-Ethylhexyl acrylate in a chronic study on male C3H mice had led to the development of papillomas and carcinomas, the present study was conducted in order to investigate whether this was an effect specific to that strain or not.

GLP compliance:

Limit test:

Species:

mouse

Strain:

other: C3H and NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany and Jackson Laboratories, Bar Harbor, USA
- Age at study initiation: 6 weeks
- Housing: single
- Diet (ad libitum): Standard-Haltungsdiaet Nr . 1324 der Fa . Altromin, Lage/Lippe, BRD
- Water (ad libitum): tap water
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1 °C
- Humidity (%): 59 ± 9
- Photoperiod (hrs dark / hrs light): 12 h light : 12 h dark

Type of coverage:

open

Vehicle:

acetone

Details on exposure:

Route of Administration: dermal

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test solutions were prepared once per month. They were analysed at the start and end of the treatment period by gas chromatography.

Duration of treatment / exposure:

3 months

Frequency of treatment:

3 times a week

Dose / conc.:

4.5 mg/cm² per day (nominal)

Remarks:

21.0 % w/w

Dose / conc.:

19.2 mg/cm² per day (nominal)

Remarks:

86.5 % w/w

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: none

Positive control:

Observations and examinations performed and frequency:

Body weight was determined before test start and then once weekly. Mortality, appearance and behavioural abnormalities were checked twice on workdays and once daily on weekends. Clinical symptoms of intoxication were recorded once daily. Skin changes at the application sites were recorded in detail.

Sacrifice and pathology:

At the end of the 3-month test period all surviving animals were sacrificed and subjected to gross-pathological examination. Animals that had died during the course of the study were also necropsied. The skin areas from the application site and organs which showed abnormalities were fixated in 10 % formalin. Samples from 5 animals/group were examined histopathologically. Three microscopical slices were prepared from every application area and stained with hematoxylin-eosin.

Statistics:

Due to the small number of animals scheduled for histopathlogical examination, no statistical evaluation was performed.

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no significant differences in body weight development between control and treatment groups. Concerning general appearance and behaviour there were no abnormalities noted amongst the animals of the treatment groups.
One C3H mouse died during the 7th week of treatment. This death was probably not test substance-related. All other animals survived until the end of the study.

BODY WEIGHT AND WEIGHT GAIN
No abnormalities were reported as compared to the historical control.

GROSS PATHOLOGY (macroscopic skin reactions)
The NMRI mice treated with 21 and 86.5 % solutions of the test material in acetone did not show any skin changes or abnormalities at the application site, except for two high dose animals with slight erythema. In contrast, the C3H mice that had been treated with a 86.5 % solution of the test material in acetone showed eschar formation at the application sites already during the 2nd week of treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
Due to the skin changes that had been observed on 10/10 C3H mice treated with the high dose, the application site of all 10 animals were examined histopathologically. Eschar formation, condensation of the subcutis, and pigment loaded hair follicles were recorded. Epidermal hyperplasia was found in 4/10 C3H mice. The 5 high dose NMRI mice which had also been subjected to necropsy and histopathological examination, showed condensation of the subcutis, but only in one animal eschar formation at the application sites. No epidermal hyperplasia were found. The skin samples of the 5 low dose NMRI mice and the 5 control mice did not reveal any abnormalities upon histopathological examination.

Dose descriptor:

NOAEL

Effect level:

4.5 mg/cm² per day (nominal)

Sex:

male

Basis for effect level:

other: Local skin effects

Critical effects observed:

not specified

Executive summary:

In a study on the skin effects of 2-EHA on two mice strains after 3-month epicutaneous application it was shown that skin irritation was more severe in C3H than in NMRI mice (BASF, 1986). 10 male C3H mice and 5 male NMRI mice were administered to 25 µl 2-EHA solution (86.5% 2-EHA in acetone) on the clipped dorsal skin (approximately 1,081 mg/kg bw/day, based on mouse body weight of 20 g) at three days per week, additionally 5 NMRI mice were treated with a 21% solution of 2-EHA in acetone (approximately 262 mg/kg bw/day), 10 male NMRI mice treated with acetone served as controls. Clinical symptoms, mortality and body growth were recorded. Macroscopic skin effects were reported from all C3H mice and five of the NMRI mice of all other groups. Histopathological examinations were restricted to the skin of the application area and of tissues with macroscopic abnormalies of 5 animals of each group.

No other clinical abnormalities other than crust formation in 10/10 C3H mice and reddening in 2/5 NMRI mice at the application site at 2-EHA concentration of 86.5% were observed. Epidermal hyperplasia was found in some C3H mice but not in the NMRI strain at 2-EHA concentrations of 86.5%. A condensation of the subcutis was reported for both strain at this concentration. No clinical or microscopic lesions were observed in NMRI mice treated with 21% 2-EHA and in vehicle control mice. With respect to local effects on the skin, a NOAEL of 25 µl of a solution containing 21% 2-EHA in acetone (170 mg/kg bw/day) administered on three days/week during 3 months was delivered for the NMRI mice. No conclusion on a systemic NOAEL can be drawn from this study.

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 4.5 mg/cm²
Study duration:: subchronic
Species:: mouse

Additional information

Repeated dose toxicity: Inhalation

In detail, degeneration of the olfactory mucosa was diagnosed in the anterior part of the nasal turbinates (level 1) in all high dose rats and in four males and four females of the mid dose group. At the high dose level, the degeneration affected the olfactory mucosa diffusely in the dorsal and dorsolateral area, and the severity was mainly moderate. Mid dose animals showed small areas of degeneration of the dorsolateral olfactory mucosa of minimal severity. At level 2 of the turbinates, degeneration was diagnosed in all high dose rats, one mid dose male, two female and one female of the low dose group, and in one control group male. In the high dose group, the degeneration was diffuse in the dorsal and dorsolateral region, whereas in the other groups the degeneration was focal. The severity was minimal to marked in the high dose group, and mainly minimal in the other groups. Slight degeneration of the olfactory mucosa of the level3 was only diagnosed in one male and one female each of the high dose group. 2-EHA induced no lesions of the trachea and the lungs, data of the pharynx/larynx were not available.

Table. 2-EHA induced olfactory degeneration in rats from a 90-day inhalation study (BASF, 1989)

Dose group	Control	Control	10 ppm	10 ppm	30 ppm	30 ppm	100 ppm	100 ppm
Sex	M	F	M	F	M	F	M	F
No. of animals	10	10	10	10	10	10	10	10
Nasal cavity Level 1 (anterior)					4	4	10	10
Mean severity grade					1	1	2.9	3.1
Level 2	1		2	1	1		10	10
Mean severity grade	1		1	1	2		2.9	2.5
Level 3							1	1
Mean severity grade							2	2

Grading used 1 = minimal, 2 = slight, 3 = moderate, 4 = marked; M = male, F = female

Reduced body weight gain, lower levels of parameters of the protein metabolism, the reduced serum glucose concentration and the reduced lipid accumulation in liver cells were assumed to be induced by a lower food consumption possibly resulting from the irritation effect on the respiratory tract of exposed animals. Similar findings were reported from repeated dose inhalation studies on acrylic acid (BASF, 1987). Although this study did not include food consumption measurement to verify this assumption, the above mentioned effects were not considered to represent relevant toxic effects. In high dose groups, a minimal liver damage was indicated by elevated activities of transaminase and alkaline phosphatase. This effect was considered to be the only systemic one of toxicological significance. In conclusion, the NOAEC for local effects on the respiratory tract was considered at 10 ppm (0.075 mg/l), whereas the NOAEC for systemic toxic effects was 30 ppm (0.225 mg/l).

Repeated dose toxicity: Oral

Stability, correct concentrations and homogeneous distribution of 2-Ethylhexyl acrylate in the diet were all confirmed through analytics.

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the test compound of 12500 ppm in F0 as well as F1A rats of both sexes.

Regarding pathology, target organs were the glandular stomach and the kidneys.

Repeated dose toxicity: Dermal

In a well-documented study on the skin effects of 2-EHA on two mice strains after 3-month epicutaneous application it was demonstrated that skin irritation was more severe in C3H than in NMRI mice (Advisory Board for Preventive Medicine and Environmental Protection Ltd., 1986). Ten male C3H mice and 5 male NMRI mice were administered 25 µl 2-EHA solution (86.5 % 2-EHA in acetone) on the clipped dorsal skin (ca. 19.2 mg/cm² or 758 mg/kg bw/day, based on an estimated treated surface of 1 cm² and mouse body weight of 25 g) at three days per week, additionally 5 NMRI mice were treated with a 21 % solution of 2-EHA in acetone (ca. 4.5 mg/cm² or 170 mg/kg bw/day), 10 male NMRI mice treated with acetone served as controls. Clinical symptoms, mortality and body growth were recorded. Macroscopic skin effects were reported from all C3H mice and five of the NMRI mice of all other groups. Histopathological examinations were restricted to the skin of the application area and of tissues with macroscopic abnormalities of 5 animals of each group.

No clinical abnormalities other than crust formation in 10/10 C3H mice and reddening in 2/5 NMRI mice at the application site at 2-EHA concentration of 86.5 % were observed.

Epidermal hyperplasia was found in some C3H mice but not in the NMRI strain at 2-EHA concentrations of 86.5 %. A condensation of the subcutis was reported for both strain at this concentration. No clinical or microscopic lesions were observed in NMRI mice treated with 21 % 2-EHA and in vehicle control mice. With respect to local effects on the skin, a NOAEL of 25 µl of a solution containing 21 % 2-EHA in acetone (170 mg/kg bw/day) administered on three days/week during 3 months was delivered for the NMRI mice. No conclusion on a systemic NOAEL can be drawn from this study.

Chronic irritative skin damage was noted in mice of a carcinogenicity study treated by dermal application of 2-EHA in acetone on weekly clipped interscapular region (BASF AG 1986; Wenzel-Hartung et al., 1989). No data on the size of treated area were reported, 10 % of the total body surface area can be used as a default assumption. Groups of 80 C3H/HeJ mice received 25 µl 2-EHA solution with 86.5 %; 43 %; 21 % and 2.5 % (w/w) in acetone (corresponding to approximately 937; 444; 212 and 24.8 mg/kg bw, calculation basis: density of test substance (0.88 g/mL) and acetone (0.79 g/mL); 20 g bw at study begin) on 3 times/week during life time or served as controls (untreated control and vehicle group). The group with a 43 % solution was treated during 24 weeks and was observed until end of life (stop-test).

Beginning within the first few weeks scaling, and/or scabbing were observed at all dose levels. Lesions observed in animals treated with 2.5 % showed a trend to regression after weeks 4 and 5 of treatment. Whereas regression of the skins lesions occurred within 7 weeks after termination of the treatment with 43 % 2-EHA solution, further skin lesions developed in the 21 % and 86.5 % groups. (see also Section 7.7 of the Technical IUCLID 5 Dossier).

In a recent publication, Murphy et al., (2018) applied contemporary evaluation criteria to the existing dermal carcinogenicity dataset. Their evaluation concluded that 2-EHA exposure resulted in chronic irritation, leading to in¿ammation, tissue injury, and wound repair, the latter of which is disrupted in C3H/HeJ mice. Using current guidance for setting the maximum tolerated dose (MTD), the 2.5% dose in the C3H/HeJ mouse met the criteria for the MTD. The dose ranges in both the C3H/HeJ and NMRI studies exceeded the MTD for skin integrity based on interim and terminal evaluations of the application site skin condition. The MTD for dermal application was clearly exceeded at dose levels >21% in C3H/HeJ mice, and met or exceeded at this concentration in NMRI mice.

Justification for classification or non-classification

GHS classification (GHS UN REV.9, 2021) identical to REGULATION (EC) No 1272/2008: no classification

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

	Test group 01 (1500 ppm)	Test group 02 (5000 ppm)	Test group 03 (12500 ppm)
F0 males	96.4	313.2	794.3
F0 females (premating)	124.8	415.8	1048.7
F0 females -lactation period -gestation period	101.6 146.9	340.3 495.6	825.2 1192.4

F0	Male animals				Female animals
Test group (ppm)	0	1500	5000	12500	0	1500	5000	12500
Glandular stomach Erosion/ulcer	0	2	4	0	3	1	1	8
Kidneys Tubules basophilic Grade 1 Grade2	5 4 1	6 5 1	8 6 2	12 10 2	5 5 0			2 2 0

Registration Dossier

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Diss Factsheets

2-ethylhexyl acrylate

Endpoint summary

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

Endpoint conclusion

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint conclusion

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint conclusion

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint conclusion

Additional information

Justification for classification or non-classification

Referenceopen all close all