2-(dimethylamino)ethyl acrylate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals; Proposal for updating Guideline 414: Prenatal Developmental Toxicity Study (22 Jan 2001)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Corrigendum to EC Commission Directive 2004/73/EC, Part B: Methods for the determination of toxicity: Prenatal Developmental Toxicity Study; Official Journal of the European Union, No. L 216, pp. 227-235 (29 Apr 2004)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methyl acrylate

EC Number:

202-500-6

EC Name:

Methyl acrylate

Cas Number:

96-33-3

IUPAC Name:

methyl acrylate

Details on test material:

- Name of test substance: Methyl Acrylate (MA)
- CAS No.: 96-33-3
- Purity: 99.9 area-%
- Physical state/Appearance: Liquid /colorless, clear
- Homogeneity: Given
- Batch No.: 010653eda0 Verkaufstank 02 vom 20.11.2007
- Storage conditions: Refrigerator (KS), avoid temperatures >35 °C, under light exclusion, light sensitive
- Stability: Expiry date: 27 Oct 2008

Test animals

Species:

rabbit

Strain:

Himalayan

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: 16-24 weeks
- Weight at study initiation (pregnant animals): 2202-2829 g
- Housing: Singly in type 12.2395.C stainless steel wire mesh cages. During exposure, the animals were kept singly in special mesh cages (40x13x16cm) from BASF SE.
- Diet: Pelleted “Kliba maintenance diet for rabbit & guinea pig, GLP” (Provimi Kliba SA, Kaiseraugst, Switzerland) ad libitum. During exposure, no food was supplied.
- Water: Tap water ad libitum. During exposure, no drinking water was supplied.
- Acclimation period: approx. 7-14 days


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

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: conditioned supply air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: glass steel inhalation chamber, volume of 1.4 m³ (BASF SE)
- Method of holding animals in test chamber: animals were kept singly in wire cages located in the inhalation chamber
- System of generating inhalation atmospheres: Generator systems: two-component atomizers (BASF SE); continuous infusion pumps PERFUSOR (B. Braun); Glass mixing stages (BASF SE); glass vaporizers with thermostat (BASF SE). Generation procedure: The test substance was used unchanged. For each concentration, the test substance was supplied to the two-component atomizer of a thermostated vaporizer at a constant rate by means of the piston metering pump. The vapor was generated by spraying the substance with compressed air into a counter current of conditioned supply air (about 50% ± 20% relative humidity, 22°C ± 2°C). Thereafter it was further mixed with conditioned supply air and passed into the inhalation system.


TEST ATMOSPHERE
- Brief description of analytical method used: GC analysis of absorption samples
- Samples taken from breathing zone: yes, immediately adjacent to the animals' noses

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Calculation of nominal concentrations: The nominal concentration was calculated from the study means of the test pump rates and the supply air flows used during exposure to generate the respective concentrations.
Online monitoring of the atmospheric concentration: The constancy of concentrations in the inhalation atmospheres were monitored continuously by calibrated online total hydrocarbon analyzer (TESTA FID 2010T in test group 0 and TESTA FID 123 in test groups 1-3) in all test groups including control. Using line recorders the measurements were recorded (see examples in Figure 003 in the APPENDIX) and the signals were transferred to the automated measuring system. The long-term drift of the online devices were checked by a weekly measurement of certificated test gas. To control the correctness of the calibrated devices and the identity of the test substance in the atmosphere, weekly two absorption samples per concentration were drawn from the atmospheres and were analyzed by gas chromatography (GC). The measured values of test group 0 served as blind control. The blind values were mainly the animals' gas emission and, in minor amount, directly from the atmosphere. Therefore, the concurrent blind values were substracted from the measured values. From the corrected daily mean values of each concentration, mean concentrations and standard deviations for the entire study were calculated.

Details on mating procedure:

- Impregnation procedure: artificial insemination
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy
- Any other deviations from standard protocol:

Duration of treatment / exposure:

6 hours on workdays over a time period of 23 consecutive days (gestation days (GD) 6–28)

Frequency of treatment:

daily on workdays

Duration of test:

Until GD 29

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 5, 15, 45 ppm
Basis:
other: target concetration

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 17.6, 52.8, 158.4 mg/m³
Basis:
other: target concentration

Doses / concentrations 3

Remarks:

Doses / Concentrations:
4.9, 15.7, 44.2 ppm
Basis:
analytical conc.

Doses / concentrations 4

Remarks:

Doses / Concentrations:
17.4, 55.3, 155.6 mg/m³
Basis:
analytical conc.

Doses / concentrations 5

Remarks:

Doses / Concentrations:
21.9, 57.2, 176.5 mg/m³
Basis:
nominal conc.

No. of animals per sex per dose:

25 inseminated female Himalayan rabbits

Control animals:

yes, sham-exposed

Examinations

Maternal examinations:

- Mortality
Mortality was checked in the females twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0 29).

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality was checked in the females twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0 29). During pre-flow period and on the day of necropsy the animals were examined for clinical symptoms at least once a day. During the exposure period, a clinical inspection of each animal was performed at least three times a day (before, during and after exposure). During the exposure procedure a groupwise examination was conducted.


BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on 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.
- Corrected (net) body weight gain: 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: Yes
- The food consumption was determined daily on GD 1–29. Because of the malfunction of a balance, the food consumption was not properly recorded on 24 June 2008and the values from this day were not used for the calculation of means and were specified as “not measured / no value determinable”.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: weight assessment: lungs; histopathology: nasal cavities, larynx, trachea, lungs, mediastinal lymph nodes, all gross lesions

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as: 1) live fetuses; 2) dead implantations: a) early resorptions (only decidual or placental tissues visible or according to SALEWSKI (Salewski, 1964) from uteri from apparently non pregnant animals and the empty uterus horn in the case of single horn pregnancy); b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible); c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)
- Calculations of conception rate and pre- and postimplantation losses were carried out:
The conception rate (in %) was calculated according to the following formula: (number of pregnant animals)/(number of fertilized animals) x 100
The preimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice according to the following formula: (number of corpora lutea – number of implantations)/(number of corpora lutea) x 100
The postimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice from the following formula: (number of implantations – number of live fetuses)/(number of implantations) x 100

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

Statistics:

DUNNETT-test (two-sided), FISHER'S EXACT test (one-sided), WILCOXON-test (one-sided), KRUSKAL-WALLIS test (two-sided)

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Details on maternal toxic effects:
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, corrected (net) body weight gain and summary of reproduction data. On female each of test group 0 (0 ppm), test group 2 (15 ppm), and test group 3 (45 ppm)were excluded from the above-mentioned calculations since they were not pregnant. 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).

- Mortality: There were no test substance-related or spontaneous mortalities in any group.
- Clinical symptoms: No test substance-related clinical signs or any disturbances of the general behavior were observed in any rabbit during the entire study period.
- Food consumption: The average food consumption was comparable to the control group in all test groups (5, 15 and 45 ppm) and did not show a test substance-related impairment. Differences between control rabbits and test substance-treated rabbits did not show a relation to dosing and were considered to be without biological relevance. This overall statement is true in spite of the slightly but significantly lower high-dose value on GD 23-24.
- Body weight data: The average body weights and body weight gain were comparable among control and treated groups (5, 15 and 45 ppm) during the entire study. All observable differences in the treated groups in comparison to the controls are without any biological relevance. This statement includes the statistically significatly decreased body weight gain in test group 3 on GD 9-11.
- Corrected (net) body weight gain: The results of the corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) revealed no difference of biological relevance between the test substance-treated groups and the control group. Mean carcass weights remained also unaffected by the treatment.
- Uterus weight: The mean gravid uterus weights of the animals of all test groups (5, 15 and 45 ppm) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance. Considering the fluctuations in the mean number of live fetuses/doe, they reflect the normal degree of variation for rabbits of the strain used in this study.
- Reproduction data of does: The conception rate reached 96% in test groups 0, 2 and 3 (0, 15 or 45 ppm) and 100% in test group 1 (5 ppm). Importantly, a sufficient number of pregnant females was available for the purpose of the study, as 24-25 pregnant rabbits per group had implantation sites in the uterus. There were no test substance related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. Generally, gestational parameters in the various test groups were within the normal range for animals of this strain and age, see also Part III (Supplement) for historical control data.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

15 ppm

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

45 ppm

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
- Sex distribution of fetuses: The sex distribution of the fetuses in test groups 1-3 (5, 15 and 45 ppm) was comparable to the control fetuses. Observable differences were without biological relevance.
- Weight of placentae: The mean placental weights in test groups 1, 2 and 3 (5, 15 and 45 ppm) were comparable to the controls.
- Weight of fetuses: The mean fetal weights of all treated groups were not influenced by the test substance. Neither female nor male fetal weights showed statistically significant or biologically relevant differences between the test substance-treated groups and the controls.
- Fetal external malformations: Three external malformations, which were associated with corresponding soft tissue and skeletal malformations, were recorded for single fetuses of test groups 1 and 2 (5 or 15 ppm). Because a dose-response relationship was missing these findings were considered to be spontaneous in nature.
- Fetal external variations: One external variation (paw hyperflexion) occurred in single fetuses of all treated groups and the control. The incidences did not demonstrate a dose-response relationship and were comparable to the historical control data. Thus an association of this finding to the treatment is not assumed.
- Fetal external unclassified observations: One unclassified external observation, i.e. blood coagulum around placenta, was recorded for one fetus of the control group and was spontaneous in nature.
- Fetal soft tissue malformations: The examination of the soft tissues revealed three malformations in single fetuses of all treated groups (5, 15 and 45 ppm). Although no findings were observed in the control and the rate of affected fetuses per litter was significantly higher in the low- and mid-dose groups, no dose related increase of the incidence of soft tissue malformations was noted. Apart from the single incidental case of small cerebrum the findings occurred at incidences comparable to the historical control data. Thus an association of the soft tissue findings to the treatment is not assumed.
- Fetal soft tissue variations: Three soft tissue variations, such as absent lung lobe (lobus inferior medialis), malpositioned carotid branch and dilated cerebral ventricle, were detected in each test group including the controls without relation to dosing. Neither statistically significant differences between the test groups nor differences to the historical control data were noted.
- Fetal soft tissue unclassified observations: Unclassified soft tissue observations, such as discolored kidney, blood coagulum around urinary bladder and hemorrhagic ovary, were recorded for some fetuses of all test groups (0, 5, 15 and 45 ppm). A relation to dosing is not present if normal biological variation is taken into account. Therefore, a test substance-induced effect is not assumed.
- Fetal skeletal malformations: Malformations of the fetal skeletons were observed in fetuses of all test groups including the controls (0, 5, 15 and 45 ppm). Neither statistically significant differences between treated groups and control nor a dose-response relationship were noted. When calculated on a fetus per litter basis, the overall incidence of skeletal malformations was comparable to the historical control data. This is also true for the severely fused sternebrae (bony plate), where the incidence of affected fetuses per litter in the high-dose group was also slightly but statistically significantly higher than the concurrent control.
- Fetal skeletal variations: For all test groups, variations were detected in different skeletal structures with or without effects on corresponding cartilaginous structures. The observed skeletal variations were related to various parts of the fetal skeleton and appeared without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data. Two isolated skeletal variations were statistically significantly higher than the concurrent and/or outside the historical control (on a fetus per litter basis). These findings are delays of ossification which is reversible and does not affect the morphology of the cervical vertebra as it becomes obvious by the unchanged underlying cartilage. Such slight retardations of the ossification process occur very frequently in gestation day 29 rabbit fetuses of this strain. Also, the increased incidences are not at all related to the dose. Thus, these findings are regarded to be of no biological relevance.
- Fetal skeletal unclassified cartilage observations: Additionally, some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all groups including the control. The observed unclassified cartilage findings did not show a relation to dosing and were considered to be spontaneous in nature.
- Summary of all classified fetal external, soft tissue and skeletal observations: Various external, soft tissue and skeletal malformations occurred throughout all test groups including the control. They did neither show a consistent pattern since a number of morphological structures of different ontogenic origin were affected nor a clear dose-response relationship. Furthermore, the overall incidences were comparable to the historical control data. Thus, non of the malformations was considered to be related to the treatment. One external (paw hyperflexion), three soft tissue (absent lobus inferior medialis, malpositioned carotid branch and dilated cerebral ventricle) and a broad range of skeletal variations occurred in all test groups including the controls. All fetal and litter incidences for these variations and the corresponding mean percentages of affected fetuses/litter did not show a relation to dosing. In addition, they can be found at a comparable frequency in the historical control data. Therefore, they were not considered to be related to the treatment.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

- Study means and standard deviations of test substance concentrations:

Test group	Target concentration		Measured concentration (FID)				Nominal concentration (mg/m³)	Effectiveness of vapor generation (%)
			(ppm)		(mg/m³)
	ppm	mg/m³	Mean	SD	Mean	SD
0	0	0	-	-	-	-	-	-
1	5	17.6	4.9	1.1	17.4	3.9	21.9	79.5
2	15	52.8	15.7	1.8	55.3	6.2	57.2	96.8
3	45	158.4	44.2	1.2	155.6	4.3	176.5	88.2

- = not measured

- Occurrence of statistically significantly increased fetal skeletal variation (expressed as mean percentage of affected fetuses/litter):

Finding	Test group 0 0 ppm	Test group 1 5 ppm	Test group 2 15 ppm	Test group 3 45 ppm	HCD Mean % (range)
Incomplete ossification of cervical centrum; unchanged cartilage	1.9	8.9**	4.9	6.1	2.2 (0.0–5.0)
Incomplete ossification of sacral arch; cartilage present	0.0	0.7	1.9*	1.2	0.3 (0.0–2.2)

ppm = parts per million; HCD = Historical control data

* = p≤0.05, ** = p≤0.01 (Wilcoxon-Test [one-sided])

- Total fetal malformations: 

		Test group 0 0 ppm	Test group 1 5 ppm	Test group 2 15 ppm	Test group 3 45 ppm
Litter Fetuses	N N	24 144	25 171	24 150	24 136
Fetal incidence	N (%)	3 (2.1%)	8 (4.7%)	7 (4.7%)	6 (4.4%)
Litter incidence	N (%)	3 (13%)	6 (24%)	6 (25%)	6 (25%)
Affected fetuses/ litter	Mean%	1.9	4.0	4.6	3.7

ppm = parts per million; N = number; % = per cent

- Total fetal variations:

		Test group 0 0 ppm	Test group 1 5 ppm	Test group 2 15 ppm	Test group 3 45 ppm
Litter Fetuses	N N	24 144	25 171	24 150	24 136
Fetal incidence	N (%)	96 (67%)	123 (72%)	119 (79%)	83 (61%)
Litter incidence	N (%)	24 (100%)	25 (100%)	24 (100%)	23 (96%)
Affected fetuses/litter	Mean%	68.8	71.9	77.9	60.5

ppm = parts per million; N = number; % = per cent

Applicant's summary and conclusion

Conclusions:

The administration of Methyl Acrylate (MA) to pregnant Himalayan rabbits via inhalation had no adverse effect on prenatal development of offspring at any of the dose levels tested (5, 15 and 45 ppm). The NOAEL for maternal toxicity is 15 ppm. The NOAEL for prenatal developmental Toxicity is 45 ppm. No adverse fetal findings of toxicological relevance were evident at any dose.

Executive summary:

The study was performed according to the proposal for updating OECD Guideline 414: Prenatal Developmental Toxicity Study (22 Jan 2001) in compliance with GLP.

Methyl Acrylate (MA) was administered to pregnant Himalayan rabbits via inhalation from implantation to one day prior to the expected day of parturition (GD 6-28).

There were no test substance-related effects on the does concerning food consumption, gross/net body weight, gestational parameters, uterine, placental and lung weights, as well as necropsy observations up to and including a dose of 45 ppm. The test substance caused a severe degeneration and atrophy of the olfactory epithelium at at least one focal area in the nasal cavity (distal levels III and/or IV) at the high-dose level (45 ppm). Though being local effects such massive findings in the respiratory tract are likely to cause a considerable amount of distress in the affected maternal animals. Since distress is supposed to influence maternal homeostasis this is considered to be a significant adverse effect on maternal organism.

Fetal examinations revealed no influence of the test compound on sex distribution of the fetuses and fetal body weights. Methyl Acrylate (MA) had no adverse effect on prenatal development of offspring at any of the dose levels tested (5, 15 and 45 ppm).

Conclusion: The NOAEL for maternal toxicity is 15 ppm. The NOAEL for prenatal developmental Toxicity is 45 ppm. No adverse fetal findings of toxicological relevance were evident at any dose.