2-(dimethylamino)ethyl acrylate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-(dimethylamino)ethyl acrylate

EC Number:

219-460-0

EC Name:

2-(dimethylamino)ethyl acrylate

Cas Number:

2439-35-2

Molecular formula:

C7H13NO2

IUPAC Name:

2-(dimethylamino)ethyl prop-2-enoate

Details on test material:

- Purity: 99.9%
- Supplier: Atofina SA Paris la Défense
- Lot No.: 004925
- Physical state: liquid
- Storage condition of test material: at +4°C, protected from light

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River France (Saint Aubin-lès-Elbeuf, France)
- Age at study initiation: 6-7 weeks
- Weight at study initiation: mean body weight 214 g (male) and 180 g (female)
- Housing: Two rats per cage of the same sex and group in wire-mesh cages (43.0 x 21.5 x 18.0 cm), except for one male and one female supplementary animals which were housed singly
- Diet: A04 C pelleted maintenance diet (U.A.R., Villemoisson-sur-Orge, France) ad libitum
- Water: filtered tap water ad libitum
- Acclimation period: at least 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2
- Humidity (%): 50±20
- Air changes (per hr): approx. 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The TS was prepared as a dispersion in the vehicle: the TS was carefully mixed with the required quantity of vehicle in order to achieve the concentration of 16.67 mg/ml and then mixed using a magnetic stir-bar. The 0.667 and 3.333 mg/ml preparations were made by direct dilution of the 16.67 mg/ml preparation. On days 1 and 2, the preparations were made daily. From day 3 until the end of the study, the preparations were made for up to seven days of treatment, according to the demonstrated stability (nine days). Preparations wrere stored at +4°C, away from light, pending utilization. The preparations were delivered each day to the animal room, protected from light, and were maintained on ice (to avoid possible evaporation) and under continuous stirring, before and during administration to the animals.

- Administration volume: 3 ml/kg/day
- Concentration in vehicle: 0, 0.667, 3.333, 16.67 mg/ml

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of samples of each preparation (including the control) prepared for use in weeks 1, 4, 8 and 13 was determined.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily.

Doses / concentrations

Remarks:

Doses / Concentrations:
2, 10 and 50 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Vehicle control: 20 males and 20 females
2 mg/kg/day: 10 males and 10 females
10 mg/kg/day: 10 males and 10 females
50 mg/kg/day: 25 males and 25 females (five supplementary males and five supplementary females were added to the 50-mg/kg/day group on study day 10)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected on the basis of the results of a previously conducted two-week oral gavage preliminary study with the same TS, in which no mortalities occurred at the dose levels of 0, 5, 20 and 80 mg/kg/day, and 20 mg/kg/day was considered to be the NOAEL.
- Post-exposure recovery period in satellite groups: 4 weeks

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FREQUENCY:
- Clinical signs: at least once a day
- Mortality: at least twice a day (treatment period), or once a day (recovery period)

BODY WEIGHT:
Once before allocation of the animals into groups, on the first day of treatment and then once a week until the end of the study.

FOOD CONSUMPTION:
Once a week, over generally a seven-day period.

FOOD EFFICIENCY:
- Efficiency of food utilisation was estimated by calculation of food conversion ratios on a weekly basis using the consumption and body weight gain data.

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: Once before the beginning of the treatment period and on one occasion in weeks 13 and 17 (recovery period).
- Dose groups that were examined: control and high dose-level groups

HAEMATOLOGY:
- Time schedule for collection of blood: during week 13 and at the end of the recovery period (week 17)
- Anaesthetic used for blood collection: Yes, light ether anaesthesia
- Animals fasted: Yes, for an overnight period of at least 14 hours
- How many animals: all surviving animals from groups 2, 3 and 4, and for the 10 animals of the control group which were assigned to the recovery group. In addition, these analyses were performed on all surviving animals of the recovery groups (animals from groups 1 and 4).
- Parameters examined: erythrocytes (RBC), haemoglobin (HB), mean cell volume (MCV), packed cell volume (PCV), mean cell haemoglobin concentration (MCHC), mean cell haemoglobin (MCH), thrombocytes (PLAT), leucocytes (WBC), differential white cell count with cell morphology (neutrophils (N), eosinophils (E), basophils (B), lymphocytes (L), monocytes (M)), reticulocytes (RETIC), prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB).

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: during week 13 and at the end of the recovery period (week 17, calcium and total cholesterol only)
- Animals fasted: Yes, for an overnight period of at least 14 hours
- How many animals: all surviving animals from groups 2, 3 and 4, and for the 10 animals of the control group which were assigned to the recovery group. In addition, the calcium and total cholesterol were measured on all surviving animals of the recovery groups (animals from groups 1 and 4).
- Parameters examined: sodium (Na+), potassium (K+), chloride (Cl-), calcium (Ca++), inorganic phosphorus (I.PHOS), magnesium (Mg++), glucose (GLUC), urea (UREA), creatinine (CREAT), total bilirubin (TOT.BIL), total proteins (PROT), albumin (ALB) albumin/globulin ratio (A/G), cholesterol (CHOL), triglycerides (TRIG), alkaline phosphatase (ALP), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), gamma-glutamyl transferase (GGT), sorbitol dehydrogenase (SDH).

URINALYSIS:
- Time schedule for collection of urine: during week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, for an overnight period of at least 14 hours
- How many animals: all surviving animals from groups 2, 3 and 4, and for the 10 animals of the control group which were assigned to the recovery group.
- Parameters examined: volume (VOLUME), pH (pH), specific gravity (SP.GRAV), proteins (PROT), glucose (GLUC), ketones (CETO), bilirubin (BILI), nitrites (NITR), blood (BLOOD), urobilinogen (UROB), cytology of sediment (leucocytes (WBC), erythrocytes (RBC), cylinders (CYLIN), magnesium ammonium phosphate crystals (AMM.PH), calcium phosphate crystals (CAL.PH), calcium oxalate crystals (CAL.OX.), cells (CELLS)), appearance (APP), colour (COLOUR).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Details on results:

- Mortality and time to death: A total of 22 (13 male and 9 female) animals died or were killed on humane grounds. All but one (a male) of these deaths occured during the treatment period. Signs of ill health and respiratory difficulty were observed prior to death in the mortality of animals. The cause of death, where evident, was lung lesions, which were considered to be due to direct irritation from regurgitated stomach contents.

- Clinical signs: No clinical signs related to treatment were observed in the males and females in the control group or those given 2 and 10 mg/kg/day. At 50 mg/kg/day, commencing from week 2-3, ptyalism, generally lasting to week 6/7, was observed in the majority of the animals which survived the treatment or treatment and recovery periods. In a few animals, this was accompanied by a short period(s) of loud breathing and/or less frequently signs of ill health. Ptyalism and loud breathing, in one male animal, were the only clinical signs observed in the recovery period.

- Body weight gain: Body weight and body weight gain in male animals given 2 or 10 mg/kg/day were unaffected by treatment. In females given 2 or 10 mg/kg/day and males and females given 50 mg/kg/day there was a dose-related, transient reduction in body weight gain (which was only occasionally statistically significant) from weeks 1/2 lasting up to 1-4 weeks. Thereafter body weight gain improved and overall body weight gain for the treatment and recovery periods was considered to be unaffected by treatment.

- Food consumption: Food conmsumption and effiency of food utilisation were unaffected by treatment and values, where calculable, were similar in all groups including controls throughout the treatment and recovery periods.

- Ophthalmoscopic examination: There were no treatment-related ophthalmic lesions detected during the examinations performed at the end of the treatment and recovery periods.

- Haematology: There were no effects of treatment on any of the parameters examined in male or female animals given 2 and 10 mg/kg/day for 13 weeks. At 50 mg/kg/day, there was a statistically significant increase in neutrophil counts (males and females), and an associated decrease in lymphocyte counts (males only; not significant) in animals given this substance for 13 week. Total white cell counts were unaffected and the effect was not present, values being similar to controls, at the end of the recovery period.

- Blood biochemistry and urinalysis: There were no effects which could definitely be related to treatment with the test substance at 2, 10 or 50 mg/kg/day for 13 weeks or 13 weeks followed by a four-week recovery period.

- Organ weights: There were no changes in absolute or relative organ weights which were considered to be related to treatment with 2, 10, or 50 mg/kg/day of this substance or of toxicological significance after 13 weeks or 13 weeks followed by a four-week recovery period.

- Gross pathology: There were no effects of treatment on any of the tissues examined in male or female animals given 2 or 10 mg/kg/day for 13 weeks. At 50 mg/kg/day the following changes were considered to be related to the treatment period:
greyish foci in the mucosa of the forestomach in 11/20 males and 3/19 females, enlaragement of the pancreatic lymph nodes in 5/20 males and 6/19 females, and dilatation and/or reddish colour of the lungs in 7/20 males and 6/19 females.
None of the above findings were seen in animals killed at the end of the recovery period.

- Histopathology: At 10 mg/kg/day, hyperplasia/hyperkeratosis, oedema and inflammatory cell infiltration (all due to direct irritance of this substance) of the forestomach submucosa were seen in some males. At 50 mg/kg/day, the following findings, considered to be treatment-related, were seen in decedents and surviving animals after 13 weeks of treatment:
ulceration, hyperplasia/hyperkeratosis, inflammatory cell infiltration and/or granulation tissue formation in the submucosa, oedema in the
mucosa and submucosa, and necrosis of the mucosa/submucosa in the forestomach. These findings were considered to be a direct irritant effect ofthe test substance on the mucosa of the forestomach.
Furthermore, alveolar haemorrhage and/or oedema and congestion in the lungs which were considered likely to be an effect of regurgitation of stomach contents.

Effect levels

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: histopathological findings in the forestomach

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Histopathological findings in the forestomach of male and female animals:

Dose group (mg/kg/day)	0 (Control)	2	10	50
Males
No. of animals	10	not tested	10	20
Oedema in mucosa
Grade 1	0		0	4
Grade 2	0		0	2
Oedema in submucosa
Grade 1	0		2	0
Grade 2	0		1	3
Grade 3	0		0	2
Grade 4	0		0	1
Granulation tissue formation in submucosa
Grade 1	0		0	2
Grade 2	0		0	3
Grade 3	0		0	1
Inflammatory cell infiltration
Grade 1	0		1	2
Grade 2	0		0	5
Grade 3	0		0	2
Ulceration
Grade 1	0		0	2
Grade 2	0		0	4
Grade 3	0		0	1
Hyperplasia
Grade 1	0		2	2
Grade 2	0		0	4
Grade 3	0		0	6
Hyperkeratosis
Grade 1	0		2	3
Grade 2	0		0	4
Grade 3	0		0	5
Females
No. of animals	10	not tested	10	20
Oedema in mucosa
Grade 1	0		0	4
Grade 2	0		0	2
Oedema in submucosa
Grade 1	0		0	3
Grade 2	1		1	3
Grade 3	0		0	3
Grade 4	0		0	0
Granulation tissue formation in submucosa
Grade 1	0		0	2
Grade 2	0		0	6
Grade 3	0		0	0
Inflammatory cell infiltration
Grade 1	0		0	5
Grade 2	0		0	5
Grade 3	0		0	1
Ulceration
Grade 1	0		0	1
Grade 2	0		0	1
Grade 3	0		0	1
Hyperplasia
Grade 1	0		1	0
Grade 2	0		0	2
Grade 3	0		0	11
Hyperkeratosis
Grade 1	0		1	0
Grade 2	0		0	3
Grade 3	0		0	10

grade 1 = minimal, grade 2 = slight, grade 3 = moderate, grade 4 = marked

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions chosen, the NOAEL of the TS when administered daily by oral gavage to male and female Sprague-Dawley rats for 13 weeks is considered to be 10 mg/kg/day.

Executive summary:

The study was conducted according to OECD guideline 408 under GLP conditions.

The TS was administered daily by oral gavage to four groups of Sprague-Dawley rats at dose-levels of 0 (control), 2, 10 and 50 mg/kg/day for 13 weeks, followed by a four-week treatment-free period in satellite groups.

The TS was clinically well tolerated at 2 and 10 mg/kg/day at the latter dose-level, treatment-related lesions (which were not associated with any adverse clinical signs) were found in the forestomach of some males. At 50 mg/kg/day, a high incidence (13 males and 9 females) of mortality was observed. Before death, respiratory difficulties and sign of ill health were observed. In surviving animals of this dose-level, ptyalism and periods of loud breathing were the main signs noted, together with increased neutrophil counts at the end of the treatment period. At histopathological investigations, substance-related lesions in the lungs and forestomach were found for both decedent animals and survivors.

Conclusion: Under the experimental conditions chosen, the NOAEL of the TS when administered daily by oral gavage to male and female Sprague-Dawley rats for 13 weeks is considered to be 10 mg/kg/day.