2-ethylhexyl acrylate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20.10.2021 - 24.01.2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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. However, another dossier update including the final study results and an assessment of its impact on the exposure assessment will be done as soon as possible.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 Jan 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl acrylate

EC Number:

203-080-7

EC Name:

2-ethylhexyl acrylate

Cas Number:

103-11-7

Molecular formula:

C11H20O2

IUPAC Name:

2-ethylhexyl acrylate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: 11498068E0
- Purity, including information on contaminants, isomers, etc.: 99.8 corr. area-%

Test animals

Species:

rabbit

Strain:

New Zealand White

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: 15-17 weeks
- Weight at study initiation: 3390 - 4513 g
- Fasting period before study: not applicable
- Housing: housed singly in Type 4X03B700CP cages supplied by TECNIPLAST Deutschland GmbH
- Diet (e.g. ad libitum): ad libitum (Kliba maintenance diet rabbit and guinea pig “GLP”, supplied by Granovit AG, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21
- Humidity (%): 46-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light):12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: diet

Details on exposure:

Test substance preparation and administration:
The test substance was administered daily as a homogeneous addition to the food with daily concentration adjustment on the basis of body weight and food consumption measurements (GD 6-29).
The test substance preparations were prepared three times: once before the start of the study and two times during the study.
The concentration of 2-Ethylhexyl acrylate in the diet was calculated with the following formula:
Food consumption: 80 g, 130 g or 170 g per day*
Mean body weight: 3900 g*
Depending on the daily individual body weight and daily food consumption of each rabbit, the diet with the most appropriate concentration for the individual animal was chosen each day.
target dose 50 mg/kg bw/d: 1147 ppm (assumed daily food intake of 170 grams); 1500 ppm (assumed daily food intake of 130 grams);2438 ppm (assumed daily food intake of 80 grams)
target dose 150 mg/kg bw/d: 3441/ 4500/ 7313 ppm (assumed daily food intake as specified before)
target dose 300 mg/kg bw/d: 6882/ 9000/ 14625 ppm (assumed daily food intake as specified before)

VEHICLE
Pelleted food (Pelleted maintenance diet rabbit and guinea pig “GLP”); test substance suspensions of appropriate concentrations were prepared and sprayed on the pellets. For the control food, 0.5% Sodium carboxymethyl cellulose suspension in deionized water (with 10 mg/100 mL Cremophor EL) was sprayed on the pellets.

Analytical verification of doses or concentrations:

yes

Remarks:

Samples were sent twice during the study period, for verification of the concentrations, homogeneity and stability.

Details on analytical verification of doses or concentrations:

Samples of the test substance preparations were sent to the analytical laboratory twice during the study period (at the delivery of food and at the end of the administration period) for verification of the concentrations. The samples taken for the first concentration control analysis at the delivery of food were also used to verify the homogeneity of the samples of all concentrations. The concentrations measured at the delivery of food and the end of administration period provided also information on the stability of the substance preparation, as the same preparation was used throughout the entire study period. From each formulation, three samples were taken from the preparation vessel (one from the top, middle and bottom).

Stability analysis
The concentrations measured at the delivery of food and the end of administration period corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations. Therefore, the stability of the test substance preparations over the study period was demonstrated.

Homogeneity analyses of the test substance preparations
The homogeneous distribution of the test substance in the vehicle (pelleted Kliba maintenance diet rabbit and guinea pig “GLP”) was demonstrated in nearly all samples, except samples Nos.6-8 and Nos. 24-26 which did not meet the specification limit of 5%, based on SOP’s of the test facility. This slight deviation from the acceptance criterion was, however, considered acceptable for the analysis of test substances in complex matrices like diet.

Concentration control analyses of the test substance preparations
The results of the analyses of the test substance preparations in pelleted Kliba maintenance diet rabbit and guinea pig “GLP” confirmed the correctness of the prepared concentrations.
The measured concentrations of the samples Nos. 3-5, 6-8, 9-11, 12-14, 15-17, 21-23, 24-26, 27-29, 62-64, 65-67, 68-70, 71-73, 74-76, 77-79, 80-82 and 83-85 corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations (see PART III, Supplement). The mean values for the samples Nos. 18-20 (111.6 %) and 59-61 (86.2 %) of 2-Ethylhexyl acrylate in diet did not meet the specification limit of 90% - 110% of the nominal concentrations. These slight deviations from the acceptance criterion was, however, considered acceptable for the analysis of test substances in complex matrices like diet.

Details on mating procedure:

- Impregnation procedure: artificial insemination
- The day of insemination referred as GD 0 (beginning of the study)of pregnancy

Duration of treatment / exposure:

The test substance was administered to the animals as a homogeneous addition to the food from GD 6-29.

Frequency of treatment:

daily

Duration of test:

30 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

191 (225) mg/kg bw/d (effective dose)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

119 (131) mg/kg bw/d (effective dose)

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

39 (42) mg/kg bw/d (effective dose)

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control group

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: In an initial palatability study, rabbits were exposed to target doses of 100, 300 and 1000 mg/kg bw/d (2294, 6882 and 22941 ppm) 2-Ethylhexyl acrylate in the diet. Compared to the control the average food consumption was reduced by about 21, 24 and 52 % in the low-, mid- and high-dose groups. Based on the extremely reduced food consumption at 1000 mg/kg bw/d resulting in significant weight loss, 500 mg/kg bw/d were chosen as high dose in the subsequent maternal toxicity study.
In the maternal range-finding study rabbits were exposed to target doses of 50, 150 and 500 mg/kg bw/d 2-Ethylhexyl acrylate in the diet. Because of the reduced food consumption, the average substance intake was 45, 136 and 238 mg/kg bw/d in the low-, mid- and high-dose groups. Reduction of food consumption was particularly distinct in the high-dose group and at the beginning of the exposure period, accompanied by a corresponding decrease of body weight. To countervail the circle of ever lower food consumption and higher substance concentration in the diet the animals were kept on the lowest concentration foreseen for the high-dose dose group from GD 10 for the rest of the study. This ensured a relatively stable consumption of the medicated diet but as a consequence the overall substance intake was lower than intended. In the end, the substance intake ranged between 152 and 341 mg/kg bw/d from GD 10 onwards. Two does in the high-dose group aborted, one at mid pregnancy after a long period of food refusal and the other one immediately before term also after a long period of decreased food consumption. Apart from food consumption/body weight effects a slight shift in blood count as well as slightly increased adrenal weights were noted in the high-dose group. Based on these data, the following target doses were chosen for the present prenatal developmental toxicity study in New Zealand White rabbits:
50 mg/kg body weight/day: as low-dose level
150 mg/kg body weight/day: as mid-dose level
300 mg/kg body weight/day: as high-dose level
The intention of the chosen concentrations was to find a balance between a sufficiently high food intake and the achievable dose level.
- Rationale for animal assignment (if not random):
The strain was selected since historical control data is available from the test facility for New Zealand White rabbits. This specific strain has been proven to be sensitive to substances with a teratogenic potential.


Analyses confirmed the correctness of the prepared concentrations, their homogeneous distribution and the stability of the test substance in the vehicle.
The average test substance intake was 39, 119 and 191 mg/kg bw/d, based on all females proven to be pregnant during the treatment period. The reason for the discrepancy to the target doses was reduced food consumption which could not be compensated by applying the procedure described in section 2.2.3. which involved the adaptation of the test substance concentration in the diet to the actual food intake.
However, cases of reduced food consumption were rather irregularly distributed among the individual rabbits within the treatment groups. It is notable, that all females experiencing abortions or complete litter losses had very long time periods of either food refusal or distinctly reduced food consumption, which in turn had a profound influence on average test substance intake of the respective treatment group.
These were the affected animals:
Animal / Day of abortion / Average food consumption (g/animal/day) / Effective dose (test substance intake; mg/kg bw/d)
50 mg/kg bw/d:
27 / GD 26 / 2.5 / 1.0
32 / GD 20 / 2.2 / 0.9
48 / GD 18 / 3.8 / 1.6
150 mg/kg bw/d:
56 / GD 22 / 6.0 / 7.3
66 / - / 2.6 / 2.7
69 / - / 2.9 / 3.3
300 mg/kg bw/d:
76 / GD 27 / 11.7 / 26.9
80 / GD 23 / 5.7 / 7.2
81 / GD 28 / 30.8 / 68.0
83 / GD 28 / 3.7 / 9.3
96 / - / 2.4 / 5.4
Considering the very low effective dose levels in these animals, it is rather unlikely that these abortions/resorptions were caused by the test substance. They were clearly a consequence of the food refusal of these individuals which happened independent of the applied test substance concentrations in the diet, as the relatively equal distribution of the cases about the dose groups shows.
If these outliers were discounted from the calculation of test substance intake, the corresponding group means were about 42 mg/kg bw/d, 131 mg/kg bw/d and 225 mg/kg bw/d for the low-, mid- and high dose group, respectively. Corresponding to similar
values for food consumption, the difference to the target doses in the low- and mid-dose groups.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity (GD 0 through GD 29).

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated based on the obtained results.
- Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- The consumption of food was recorded daily during GD 0-29.
- The intake of test substance was calculated from the amount of food consumed and expressed in milligram per kilogram body weight per day.

POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day 29
- Organs examined:

OTHER:
- Mortality: A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-29).
- Clinical pathology: In the morning blood was taken from the ear vein from not-fasted animals without anesthesia. 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.
- Pathology:
- Necropsy: After the does have been sacrificed, they were necropsied and assessed by gross pathology. Special attention has been given to the reproductive organs. Animals Nos. 3, 27, 32, 48, 56, 76, 80, 81 and 83 died intercurrently or were sacrificed. They were necropsied and assessed by gross pathology as soon as possible after their death.
- Organ weights: The following weights were determined in all does sacrificed on schedule: 1. Adrenal glands (fixed); 2. Kidneys; 3. Liver; 4. Spleen. All paired organs were weighed together (left and right).
-Organ/tissue fixation: The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution: 1. All gross lesions; 2. Adrenal glands; 3. Kidneys; 4. Liver; 5. Spleen; 6. Stomach. No further examinations or procedures were performed in the study.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Numver of dead fetuses: Yes
After the weight of the uterus had been determined, all subsequent evaluations of the does and the gestational parameters (except of gross pathology and organ weights) were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose, animal numbers were encoded.

Blood sampling:

- Plasma: Yes
- Serum: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter (and the heads of any fetus which revealed severe findings during the external examina-tion, e.g. anophthalmia, microphthalmia or hydrocephalus)
All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.

Statistics:

Statistics of clinical, necropsy and fetal examinations:
DUNNETT-test (twosided) for the hypothesis of equal means:
Food consumptiona), body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each
litter, litter mean fetal body weight, litter mean placental weight

FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions: Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings

WILCOXON-test (one-sided) for the hypothesis of equal medians: Proportions of fetuses with
malformations, variations and/or unclassified observations in each litter

Statistics of clinical pathology:
Blood parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test. 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 (twosided) for the hypothesis of equal medians.

Statistics of pathology:
Organ weights: Non-parametric one-way analysis using the Kruskal-Wallis test (two-sided). If the resulting p-value was equal to or less than 0.05, a pair-wise comparison of each dose group with the control group was performed using the Wilcoxon test (two-sided) for the hypothesis of equal medians.
Carcass weights: Comparison of each group with the control group was performed using the Dunnett test (two-sided) for the hypothesis of equal means.

Indices:

- The conception rate (in %) was calculated according to the following formula:
(number of pregnant animals/ number of fertilized animals) x100
- The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
(number of corpora lutea – number of implantations/ number of corpora lutea) x100
- The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
(number of implantations – number of live fetuses/ number of implantations) x100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

For some does of the test groups 0-3 (0, 50, 150 or 300 mg/kg bw/d), blood in bedding was recorded: control doe No. 9 (GD 23), low-dose does Nos. 26 (GD 26), 32 (GD 19 - this female showed also hypothermia and was sacrificed after abortion on the next day) and 37 (GD 21), mid-dose does Nos. 66 (GD 14, GD 16-18), 69 (GD 12, 13, 17) and high-dose doe No. 96 (GD 17-19). Three females of the mid- and high-dose groups (Nos. 66, 69 and 96) had no viable fetuses.
In total, reduced defecation was observed in five control, 13 low-dose, 13 mid-dose and 19 high-dose females (0, 50, 150 and 300 mg/kg bw/d). No defecation was observed in three control, eight low-dose, five mid-dose and 10 high-dose females. Although frequency of defecation usually shows great variability among individual rabbits across treatment groups during a study, the higher incidence of these observations in the test substance-treated groups comes along with reduced food consumption in the affected animals. There were no further clinical findings in the other does in this study.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female of the control group (No. 3) had to be sacrificed moribund for animal welfare reasons (accidental spinal fracture) on GD 1.
Three low-dose females (50 mg/kg bw/d - Nos. 27 on GD 26, 32 on GD 20, 48 on GD 18), one mid-dose female (150 mg/kg bw/d - No. 56 on GD 22) and four high-dose females (300 mg/kg bw/d - Nos. 76 on GD 27, 80 on GD 23, 81 on GD 28, 83 on GD 28) were sacrificed after abortion ahead of schedule. The affected animals showed a particularly distinct reduction of food consumption during the days upcoming to the abortion.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The high dose (300 mg/kg bw/d) caused a body weight loss in the respective treatment group during the first week of treatment (GD 6-14), followed by a stagnation of body weight during mid pregnancy and some days of recovery shortly before the end of the study. This resulted in statistically significantly reduced body weights on GD 14-25 (up to 8% below control) and some convergence to the control values afterwards.
In the low- and mid-dose groups (50 and 150 mg/kg bw/d) the mean BWC was statistically significantly reduced only at the beginning of treatment (GD 6-9; -32.5 g* [p ≤ 0.05] and -77.4 g** [p ≤ 0.01], respectively) and was normal otherwise considering the high variability of this parameter in rabbits. Body weight was significantly lower in the mid-dose group on a single occasion (GD 16) only.
Although the differences were not statistically significant all treated groups gained less weight in comparison to the concurrent control group during the treatment period (GD 6-29).

Corrected (net) body weight gain:

Mean carcass weight of the does of test group 3 (300 mg/kg bw/d) was reduced (-5%) in comparison to the control group (without attaining statistical significance). Furthermore, the corrected body weight change (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) was impaired in the high-dose group, attaining statistical
significance (-411.1 g*) in comparison to the concurrent control group (-218.1 g).
Mean carcass weights and corrected body weight change of test groups 1 and 2 (50 and 150 mg/kg bw/d) were not significantly different in comparison to the control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In comparison to the control group the mean food consumption of the does in the substance treated groups was reduced during several parts of the treatment period, attaining statistical significance on GD 6-17 (test group 3 - 300 mg/kg bw/d), on GD 6-16 (test group 2 - 150 mg/kg bw/d) and on GD 6-7, GD 9-10 and GD 11-16 (test group 1 - 50 mg/kg bw/d).
However, these average values include all animals who experienced abortions or complete litter loss, too. As these individuals suffered from negligible food intakes over long time periods during the study, and thus skewed the group average considerably, food consumption needed to be evaluated on an individual animal basis.
Intake of test substance:
The amount of test substance (in mg) which was consumed by the animals per kilogram body weight each day was calculated at the times at which food consumption was determined during the administration period (GD 6-29). Test substance intake represents the effective average dose levels which were achieved in the present study.
Mean maternal test substance intake:
Test group 1 (50 mg/kg bw/d): 39.1 mg/kg bw/d
Test group 2 (150 mg/kg bw/d): 118.8 mg/kg bw/d
Test group 3 (300 mg/kg bw/d): 191.3 mg/kg bw/d

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed.
At study day 29, in does of test group 1 (50 mg/kg bw/d) absolute and relative neutrophil counts were significantly decreased whereas absolute and relative monocyte counts, and relative lymphocyte counts were significantly increased. Additionally, in does of test group 2 (150 mg/kg bw/d) absolute neutrophil counts were significantly decreased whereas relative monocyte counts were significantly increased. All mentioned alterations were not dose dependent.
Therefore, these changes were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.
At study day 29, in does of test group 1 (50 mg/kg bw/d) glucose levels were significantly increased, but the change was not dose dependent. Therefore, this alteration was regarded as incidental and not treatment-related.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The mean gravid uterus weights of the rabbits of test groups 1-3 (50, 150 or 300 mg/kg bw/d) were not significantly different from controls. The differences between these groups and the control group showed no dose-dependency and were assessed to be without biological relevance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Details on results:

Clinical examinations
Only pregnant does were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant does with scheduled sacrifice (GD 29) were used for the calculation of mean gravid uterine weights, mean organ weights, corrected (net) body weight gain and summary of reproduction data.
The following females were excluded from the above-mentioned calculations:
Test group 0 (0 mg/kg bw/d):
• females Nos. 2, 4, 6, 24 - not pregnant
• female No. 3 - sacrificed moribund
Test group 1 (50 mg/kg bw/d):
• females Nos. 29, 30, 40 - not pregnant
• females Nos. 27, 32, 48 - sacrificed after abortion
Test group 2 (150 mg/kg bw/d):
• female No. 63 - not pregnant
• female No. 56 - sacrificed after abortion
Test group 3 (300 mg/kg bw/d):
• females Nos. 78, 97 - not pregnant
• females Nos. 76, 80, 81, 83 - sacrificed after abortion
Thus, according to the requirements of the corresponding test guidelines, each test group including the controls contained a sufficient number of females with implantation sites at necropsy (approximately 20, but not fewer than 16 females with implantation sites).

- Absolute weights: All mean absolute weight parameters did not show significant differences when compared to the control group 0.
- Relative organ weights: All mean relative weight parameters did not show significant differences when compared to the control group 0.

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

Resorption rate (i.e. post-implantation loss) was not statistically significantly different from control in any of the treatment groups, however the mid- and high-dose values (17.0 and 16.4 mean%) were above the historical range of the test facility (2.1 – 11.3 mean%). This was due to two females of the mid-dose group (Nos. 66 and 69) and one female of the high-dose group (No. 96) which had no viable fetuses but just early resorptions in their uteri. The resulting 100% post-implantation loss in these animals contributed higher than average to the group means.
These litter losses were associated with extensive periods of food refusal by these animals and were regarded as not being related to a potential developmental toxicity of the test compound.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

Two dead fetuses from high-dose doe No. 77 are within the historical control range and therefore considered to be an incidental finding.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

conception rates were 84% / 88% / 96% / 92% in test groups 0-3 (0, 50, 150 and 300 mg/kg bw/d)

Details on maternal toxic effects:

There were no test substance-related and/or biologically relevant differences between the test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre-implantation loss and viable fetuses. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

191 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: high dose

Remarks on result:

other: prenatal developmental toxicity

2

Dose descriptor:

NOAEL

Effect level:

119 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

food consumption and compound intake

Remarks on result:

other: maternal toxicity

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean weight of the male high-dose fetuses (300 mg/kg bw/d) was slightly lower than concurrent control (-12%). Although the difference attained statistical significance, the mean male fetal weight was well within range of 95% spread of the historical control data (HCD: mean 37.1 g [24.8 - 49.4]). The mean weight of the high-dose female fetuses was comparable to the concurrent control. When both sexes were combined, the mean fetal weight of the high-dose group was also comparable to control. The mean group value (35.6 g) was close to the mean and well in the range of 95% spread of the historical control data (HCD: mean 36.8 [24.4 -49.2]). The slightly lower average weight of the high-dose fetuses can be attributed to the larger average litter size in this group compared to the control, the same effect is also noticeable in the low-dose group.
The mean fetal weights of test groups 1 and 2 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (50, 150 and 300 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- One external malformation (‘umbilical hernia’) was recorded for two control fetuses (0 mg/kg bw/d). Female fetus No. 55-01 (150 mg/kg bw/d) had multiple external malformations concerning its whole fetal body (compacted appearance, multiple bulges in the region of the neck, throat and axilla of both forelimbs) (see Attachments, Table 1).
-The distribution of external malformations about the dose groups does not indicate an association to the tre atment, no statistically significant differences between the groups were noted (see Attachments, Table 2).
- No external variations were recorded.
- One unclassified external observation was recorded. ‘Placentae necrobiotic’ was seen in one fetus of the control group and is considered as incidental (see Attachments, Table 3).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- One skeletal malformation was recorded for a single mid-dose fetus (150 mg/kg bw/d). This finding is considered to be spontaneous in origin and not treatment-related (see Attachments, Table 8 + Table 9).
Fetal skeletal variations:
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dosing. The addition of skeletal variations resulted in an increased affected fetuses per litter incidence in test group 2, attaining statistical significance. However, due to the lack of dose-response relationship and the fact, that the overall incidence of test group 2 was only slightly above the historical control range, while the other incidences of test groups 0, 1 and 3 were well within (HCD: mean% 91.7 [84.9 - 97.2]), this increase is evaluated to be spontaneous in origin and not treatment-related (see Attachments, Table 10).
Fetal skeletal unclassified cartilage observations:
Some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the sternum and the ribs and did not show any relation to dosing. Therefore, they are assessed as not treatment-related (see Attachments, Table 11).

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Soft tissue malformations occurred in all test groups including controls (0, 50, 150 or 300 mg/kg bw/d). All listed findings are part of the normal background of malformations in this rabbit strain as they are all present in the historical control data. In terms of incidences no statistically significant nor toxicologically relevant differences between the groups were noted, and the overall malformation rate was inside the historical control range (see Attachments, Table 4 + Table 5).

Details on embryotoxic / teratogenic effects:

Fetal soft tissue variations:
The examinations of the soft tissues revealed cystic dilatation of the brain, dilated aorta, malpositioned carotid branch, absent lung lobe (Lobus inferior medialis) and dilated renal pelvis in individual fetuses of the different test groups (0, 50, 150 or 300 mg/kg bw/d). No statistically significant or toxicologically relevant differences between the groups were noted, and the overall incidences were within the historical control range (see Attachments, Table 6).

Fetal soft tissue unclassified observations:
Two unclassified soft tissue observations were recorded: discolored thymus in two mid-dose fetuses and a blood coagulum around urinary bladder in fetuses of all test groups. These findings are considered to be incidental (see Attachments, Table 7).

Assessment of all fetal external, soft tissue and skeletal observations:
There were noted external, soft tissue and skeletal malformations in all substance-treated test groups (50, 150 or 300 mg/kg bw/d) as well as in the control. The distribution of total malformations about the groups was not related to dose, in fact the lowest rate was noted in the high-dose group.

 In total, five fetuses had more than one malformation. Female control fetus No. 7-06, low-dose male fetus No. 49-06 (50 mg/kg bw/d) and high-dose female fetus No. 89-05 (300 mg/kg bw/d) had malpositioned kidneys combined with short ureter, respectively. For low-dose male fetus No. 49-04 an absent subclavian, malpositioned kidney and a short ureter were recorded. Furthermore, female mid-dose fetus No. 55-01 (150 mg/kg bw/d) had multiple external
malformations concerning its whole fetal body (compacted appearance, multiple bulges in the region of the neck, throat and axilla of both forelimbs). No ontogenetic pattern is recognizable for the individual malformations nor was there a cluster of any of these individual malformations seen in the other offspring of these test groups.
Further malformations, such as umbilical hernia (test group 0), enlarged lens (test groups 1-3) and severely fused sternebrae (bony plate) (test group 2) were scattered observations in individual fetuses. All these findings are not dose-related and part of the normal background of malformations in this rabbit strain as they are all present in the historical control data. Thus, a relation to the treatment is not assumed (see Attachments, Table 12).

External variations did not occur in any fetus in this study. A spontaneous origin is assumed for the soft tissue variations and the broad range of skeletal variations which were observed in fetuses of all test groups including the controls (see Attachments, Table 13).
Although the total rate of affected fetuses per litter was statistically significantly increased in the mid-dose group (150 mg/kg bw/d), the overall incidences of all classified fetal variations were comparable to the historical control range (HCD: mean% 91.98 [86.02 - 97.16]). Due to the lack of dose-response relationship and the fact, that the overall incidence of test group 2 was only slightly above the historical control range, the increase is considered to be spontaneous in origin and not treatment-related.

A spontaneous origin is assumed for the unclassified external, the unclassified soft tissue and the unclassified skeletal cartilage observations, which were observed in several fetuses of test groups 0-3. The distribution and type of these findings do not suggest any relation to treatment. Thus, fetal examinations revealed that there is no adverse effect of the compound on the respective morphological structures up to the highest dose tested (300 mg/kg bw/d).

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

191 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: high dose

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of 2-Ethylhexyl acrylate as homogeneous inclusion in the diet to pregnant New Zealand White rabbits from implantation to the expected day of parturition (GD 6-29) caused evidence indicative of maternal toxicity (food refusal because of stomach irritation) at the highest dose. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is effectively 119 mg/kg bw/d.
Since there was no evidence for treatment-related adverse effects of the test substance on fetal morphology the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is effectively 191 mg/kg bw/d.

The test substance is considered not teratogenic in rabbits under the test conditions employed in this study.