Ethyl 4-dimethylaminobenzoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 March 1989 to 20 April 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

not precised

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

not precised

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 4-dimethylaminobenzoate

EC Number:

233-634-3

EC Name:

Ethyl 4-dimethylaminobenzoate

Cas Number:

10287-53-3

Molecular formula:

C11H15NO2

IUPAC Name:

ethyl 4-(dimethylamino)benzoate

Test material form:

solid: crystalline

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl: CD® SD BR VAF PLUS

Details on species / strain selection:

The rats used were selected on the basis of the experience with the strain. The route of administration, oral gavage, was in accordance with regulatory requirements.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River France
- Age at study initiation: 28 ± 1 days old
- Weight at study initiation: 76 to 89 grams
- Housing: 5 animals of a single sex per cage
- Diet: Biosure LAD 1 Diet (ad libitum)
- Water: ad libitum access to drinking water
- Acclimation period: A minimum of 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: Mean minimum of 20.1 to mean maximum of 20.4 °C
- Humidity: Mean minimum of 50.8 to a mean maximum of 55.0 % (relative)
- Air changes: Approximately 20 per hour
- Photoperiod: 12 hours of artificial light in each 24 hour period

IN-LIFE DATES
- From: 09 March 1989
- To: 20 April 1989

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was prepared on the day of dosing by dilution with corn oil. The house dose was initially prepared and aliquots subsequently diluted further with the vehicle to obtain the intermediate and low doses. Prior to dosing the test material formulations were mixed initially by repeated inversion (20 x) and subsequently using a magnetic stirrer for a period of at least ten minutes immediately before dosing commenced.

- Dose volume: 5 mL/kg/day

VEHICLE
- Concentration in vehicle: 18, 1.48 and 0.12 % (w/v) for the high, intermediate and low doses, respectively.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed on dosing solutions prepared on Day 1.

GAS-LIQUID CHROMATOGRAPHIC METHOD
Instrument: PU 4500 Philips
Detector: FID
Column: 3' 10 % OV-7
Column Temperature (isothermal): 220 °C
Carrier Flow Rate: 30 mL/min.
Injector Temperature: 250 °C
Detector Temperature: 250 °C
Attenuation: 64 x 10^2 for the high and intermediate dose; 8 x 10^2 for the low dose
Injection Volume: 2 µL for the high and intermediate dose; 3 µL for the low dose
Retention time: Test material 2.9 minutes; eicosane 4.8 minutes

SAMPLE PREPARATION
Accurately weigh 30.15 g of test material standard into a 10 cm3 volumetric flask. By pipette add 10 cm of 1 % Eicosane in toluene as an internal standard. For the 18.1 % in corn oil concentration weigh accurately 0.9 g into a 25 cm3 volumetric flask. By pipette add 10 cm3 of 1 % Eicosane in toluene. For the 1.48 % in corn oil concentration, prepare as above weighing accurately 10.0 g.
Accurately weigh 0.015 g of test material standard into a 10 cm3 volumetric flask. By pipette add 10 cm of 0.1 % Eicosane in toluene as an internal standard. For the 0.12 % in corn oil concentration weigh accurately 11.5 g into a 25 cm3 volumetric flask. By pipette add 10 cm3 of 0.1 % Eicosane in toluene.

RESULTS
18.1 % in corn oil concentration: Assay = 18.36 % w/w
1.48 % in corn-oil concentration: Assay = 1.57 % w/w
0.12 % in corn oil concentration: Assay = 0.128 % w/w

STABILITY TESTING
The samples were reanalysed after storing at room temperature in the dark for 160 days and again after heating to 50 °C for 5 days.

18.1 % concentration: Initial Assay = 18.36 % w/w; Assay after 160 days = 18.36 % w/w; and Assay after heating to 50 °C for 5 days = 18.25 % w/w
1.48 % concentration: Initial Assay = 1.57 % w/w; Assay after 160 days = 1.57 % w/w; and Assay after heating to 50 °C for 5 days = 1.49 % w/w
0.12 % concentration: Initial Assay = 0.12 % w/w; Assay after 160 days = 0.12 % w/w; and Assay after heating to 50 °C for 5 days = 0.105 % w/w

Duration of treatment / exposure:

28 days

Frequency of treatment:

Dosing once daily, seven days per week for 4 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

5 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dosage levels were selected on the basis of available toxicity data, in particular a preliminary study in which the test material was orally administered to a group of 3 male and 3 female rats for 7 consecutive days at 1000 mg/kg/day. Clinical signs attributable to treatment included abnormal body carriage (hunched posture) and post-dose salivation throughout the study and, less frequently, pilo-erection and greasy fur. However, there were no treatment-related changes at macroscopic autopsy. The above results suggested 900 mg/kg/day to be an appropriate high dosage level for the main study.
- Rationale for animal assignment: 5 males and 5 females were randomly allocated to each of 4 groups using a computer program, so that weight distribution within each group was similar and the initial group mean body weights were approximately equalised.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked twice daily for signs of ill-health, behavioural changes or toxicosis. Any observed changed were recorded. A check occurred twice each day for death or moribundity.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed prior to commencement of dosing and subsequently at weekly intervals throughout the study.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption for each cage was measured at weekly intervals throughout the study.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 27 from the orbital sinus
- Anaesthetic used for blood collection: Yes, light ether anaesthesia
- Animals fasted: Yes, overnight prior to collection
- How many animals: All rats
- The following parameters were examined: Packed cell volume (PCV), haemoglobin (Hb), red blood cell count (RBC), platelet count (Plts), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), total white blood cell count (WBC), differential white blood cell count (neutrophils, lymphocytes, eosinophils, basophils and monocytes) and thrombotest. Cells were also evaluated for abnormal morphology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 27 from the orbital sinus
- Anaesthetic used for blood collection: Yes, light ether anaesthesia
- Animals fasted: Yes, overnight prior to collection
- How many animals: All rats
- The following parameters were examined: Glucose, glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), alkaline phosphatase (AP), total bilirubin, cholesterol (Chol), urea nitrogen (Urea Nitr), total protein, albumin (Alb), globulin (Glob), albumin/globulin ratio (A/G), sodium (Na), potassium (K), calcium (Ca), chloride (Cl), inorganic phosphorus (P) and creatine.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
After 28 days of treatment (day 29) all animals were killed by carbon dioxide asphyxiation and a complete autopsy was undertaken. The macroscopic appearance of the tissues was recorded.
The following organs were dissected free of fat and weighed: Adrenals, kidneys, liver, ovaries and testes.

HISTOPATHOLOGY: Yes
The following tissues were preserved in 10 % buffered formalin for histopathological examination: Adrenals*, aorta, brain (medullary, cerebellar and cortical sections), caecum, colon, duodenum, eyes (Davidson's fluid as fixative), heart*, ileum, jejunum, kidneys*, larynx, liver*, lungs, lymph nodes (cervical and mesenteric), mammary gland, oesophagus, ovaries, pancreas, pharynx, pituitary, prostate, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, spleen*, sternum (for bone and marrow section), stomach, testes (including epididymides), thymus (where present), thyroid (with parathyroid), tongue, trachea, urinary bladder, uterus, vagina and any other macroscopically abnormal tissue*.
Fixed tissue samples required for microscopic examination were embedded in paraffin wax, sections cut at 4 µm and stained with haematoxylin and eosin. Microscopic examinations were carried out for all tissues marked with an asterisk for rats of Group 1 (control group) and Group 4 (high dosage group) killed on Day 29.
Examinations were extended to rats of the intermediate and low dosage groups to cover those tissues (spleen and testes) exhibiting response to treatment at the high dosage level.

Statistics:

All statistical analyses were performed separately for males and females. Body weight data were analysed using weight gains. The following sequence of statistical tests was used for bodyweight, organ weight and clinical pathology data:
If the data consisted of predominantly one particular value (relative frequency of the mode exceeds 75 %), the proportion of values different from the mode was analysed using appropriate methods. Otherwise:
Bartlett’s test was applied to test for heterogeneity of variance between treatments. Where significant (at the 1 % level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
If no significant heterogeneity was detected a one-way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by transformation, the Kruskal-Wallis analysis of ranks was used.
Analyses of variance were followed by Student’s t test and Williams’ test for a dose-related response, although only the one thought more appropriate for the response pattern observed was reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the t test and Williams’ test (Shirley’s test).
When appropriate for organ weight data, analysis of covariance was used in place of analysis of variance in the above sequence. The final bodyweight was used as covariate in an attempt to allow for differences in the bodyweight which might have influenced the organ weights.
Additional or alternative statistical methods were used where appropriate.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

A slight post-dose increase in salvation was recorded for rats treated at 6 mg/kg/day (Days 11 to 14 and/or 2 to 10) and 74 mg/kg/day (Days 2 to 14, 16, 18, 20 and/or 28). This clinical finding was also found for rats treated at 900 mg/kg/day on Days 2 to 14, 16 to 24, 26 to 28 and/or 15. Other clinical signs of reaction to treatment found amongst rats of the high dosage level were abnormal body carriage (hunched posture) on Days 7 to 28 and/ or 5 and 6 and lethargy on Days 15, 16 and/or 5 and 7 to 14. Female animals of this group also showed gasping respiration on Days 11 to 14 and 16.
There were no other signs of ill health or behavioural chances that were considered to be related to treatment. Greasy fur, observed from Day 2 or Day 10 for rats of the control and low dosage groups, respectively, or throughout the whole duration of the study (rats of the intermediate and high dosage groups) was related to the nature of the vehicle (corn oil). Pallor, observed on Day 28 in all rats, was considered to be related to the blood sampling procedure carried out on Day 27.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A trend of low bodyweight gain, attaining statistical significance (P<0.05) for Weeks 1, 2 and 4, was recorded amongst male rats of Group 4 (900 mg/kg/day) in comparison with the controls. Although a similar trend was not observed for Group 4 females, this disturbance may be indicative of a treatment related effect.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The quantity of food consumed by animals receiving the test material was similar to that of the controls.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Significantly reduced PCV, Hb and RBC (P<0.01) values were recorded for the rats treated at 900 mg/kg/day in comparison with control animals. This apparent anaemic effect, accompanied by a statistical reduction in MCHC values (males) and an increase in MCV values (females) at the high dosage group (P<0.01), can be attributed to a toxicological effect of treatment.
Slight polychromasia was also observed amongst rats at 900 mg/kg/day and this finding in conjunction with the increased MCV amongst females probably indicated an erythropoietic response to the reduction in red cell parameters in this group. One Group 4 male also showed slight anisocytosis.
Amongst animals treated at 74 mg/kg/day red cell parameters (PCV, Hb and RBC) were slightly lower than corresponding control values, but differences were not statistically significant and there were no morphological abnormalities observed from blood slides. No disturbance of red blood cell parameters was observed at 6 mg/kg/day.
Raised total white blood cell counts (P<0.05), arising from a statistically significant increase in neutrophil values (P<0.01) and a non-significant increase in lymphocyte counts, were observed for female rats of the high dosage group in comparison with the controls. An increased white cell count, due to an increase in lymphocyte count was also observed amongst males at 900 mg/kg/day although differences were not significantly different. No disturbances of white cell parameters were observed at 6 or 74 mg/kg/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The raised bilirubin values (P<0.001) found for rats of this high dosage group compared with the control levels may be treatment related.
Statistically significant increases in creatinine and chlorine values (males), globulin levels (females) and the A/G ratio (both sexes) were recorded for rats treated at 900 mg/kg/day in comparison with the respective control groups (P<0.05 or P<0.01). These changes however, were of a minor nature, all being within the normal variation for rats of this age and strain.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Greatly reduced testes weights were recorded for rats receiving 900 mg/kg/day test material in comparison with the control animals (P<0.01). Liver weight amongst females receiving 900 mg/kg/day was increased relative to controls (P<0.01).
There were no other statistically significant differences between the organ weights of rats treated with the test material and those of the controls.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Pathology revealed an increased incidence of dark-coloured spleens (10/10) and the presence of small, blue-coloured testes (5/5) for rats of the high dosage level in comparison with the controls (0/10 and 0/5, respectively). Additionally, an increased incidence of females receiving the test material at 900 mg/kg/day with apparently badly groomed dorsal fur was recorded.
A raised incidence of enlarged cervical lymph nodes was recorded amongst rats of all groups and this was attributed to chance. There were no other changes that were considered to be treatment related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

-Spleen
Minimal to moderate degrees of haemosiderosis were seen in the spleens of 5/5 male and 5/5 female rats receiving the 900 mg/kg/day dose. This change was not seen in any of the intermediate, low or control group male or female animals.
Minimal to moderate degrees of extra-medullary haemopoiesis were seen in 5/5 male and 5/5 female animals receiving 900 mg/kg/day and a minimal degree was seen in 3/5 male and 2/5 female animals receiving 74 mg/kg/day. This feature was not seen in any of the low dosage or control group animals.
Moderate numbers of prominent germinal centres in the white pulp were seen in the spleens of 4/5 males receiving 900 mg/kg/day. No high dose females showed this feature. Minimal numbers of prominent germinal centres were seen in 1/5 males at 900 mg/kg/day, in 4/5 males and 1/5 females at 74 mg/kg/day, in 1/5 males and 1/5 females at 6 mg/kg/day and in 4/5 males in the control group. The increased prominence of this change at the high dose level suggests a treatment-related exacerbation.

-Testes
A moderate degree of tubular atrophy was seen in the testes of 5/5 males receiving 900 mg/kg/day. Furthermore, 2/5 high dose males had multinucleate giant cells in the seminiferous epithelium, 4/5 high dose males had an absence of spermatozoa in the epididymides, while 2/5 high dose males had multinucleate giant cells present in this tissue. These changes were not seen in any of the intermediate or low group males.

All other histopathological changes recorded were within the background range of changes normally seen in rats of this age and strain. Consequently, they were not considered to be of any toxicological significance.
The administration of the test material at 900 mg/kg/day induced testicular atrophy in male animals. The test material also induced splenic changes in all animals receiving 900 mg/kg/day and had a marginal effect on the spleens of a proportion of animals receiving 74 mg/kg/day.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

At 900 mg/kg/day findings of toxicological significance included clinical signs, reduced weight gain, effects on red and white blood cell parameters with associated splenic changed (haemosiderosis, extramedullary haemopoiesis and prominent germinal centres in the white pulp)and testicular atrophy.
At the intermediate dosage of 74 mg/kg/day, besides salivation after dosing, there was a minor disturbance of red cell parameters with an associated minimal degree of extramedullary haemopoiesis in the spleen. Although 74 mg/kg/day cannot therefore be defined as a clear no observable level, the findings at this dosage were considered to be of minor toxicological importance.
At the low dosage level of 6 mg/kg/day only salivation after dosing was observed; this was not toxicologically important.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

haematology

histopathology: non-neoplastic

Target system / organ toxicity

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study the NOAEL of the test material was determined to be 74 mg/kg/day. Changes observed at this dose were considered to be of minor toxicological importance.

Executive summary:

The subacute repeated dose oral toxicity of the test material was investigated in accordance with the standardised guidelines OECD 407 and EU Method B7 under GLP conditions by gavage.

The test material was formulated daily as 0.12, 1.48 and 18 % (w/v) suspensions in corn oil and administered orally to Crl : CD® SD BR VAF PLUS strain rats at dosage levels of 0, 6, 74 and 900 mg/kg/day to 5 rats per sex per dose level. Animals were dosed once daily for 28 days and control animals received corn oil alone. Animals were observed for clinical signs, body weight changes and food consumption throughout the study. Prior to terminal sacrifice, animals underwent haematology and clinical chemistry evaluation. All animals were sacrificed after 28 days of exposure and subjected to necropsy, consisting of gross and histopathological examinations.

A slight post-dose increase in salivation was recorded for rats of all treatment groups. This was accompanied in rats treated at 900 mg/kg/day by abnormal body carriage (hunched posture), lethargy and, in the case of the females, gasping respiration.

Bodyweight gains for the male rats receiving 900 mg/kg/day were lower than those of the male controls throughout the study with a statistical reduction (P<0.05) occurring at weeks 1, 2 and 4. This finding was considered to be treatment related.

A significant effect on red cell parameters, namely reduced PCV, Hb and RBC values (both sexes), and reduced MCHC values (males) were recorded for rats receiving 900 mg/kg/day in comparison with the controls (P<0.01 on all occasions). This impairment on red cell parameters was accompanied by increased (P<0.01) MCV (females) and slight polychromasia suggesting a possible erythropoietic response. Raised total white blood cell counts were also observed at 900 mg/kg/day arising from increased lymphocyte (both sexes) and increased neutrophil (females only) counts. A lesser effect on red cell parameters was observed at 74 mg/kg/day; however, differences from control values were not statistically significant and were considered of minor importance. Haematological parameters at 6 mg/kg/day were unaffected.

Significantly raised bilirubin values (P<0.001) were observed for rats at 900 mg/kg/day in comparison to the control levels. Increased creatinine and chloride values (males), increases in globulin levels (females) and the A/G ratio (both sexes) recorded for the rats receiving 900 mg/kg/day (P< 0.05 or 0.01) were considered to be of minor importance with all values being within the natural variation found for rats of this age and strain. No biochemical disturbance was observed amongst rats receiving 6 or 74 mg/kg/day.

A statistically significant reduction (P<0.01) in testes weight was recorded for rats receiving 900 mg/kg/day; female rats treated at this dosage showed increased liver weight (P<0.01).

Macroscopic findings after 28 days of treatment included an increased incidence of dark-coloured spleens and the presence of small blue-coloured testes for rats at 900 mg/kg/day. The appearance of apparently badly groomed dorsal fur was recorded for the females of this group. There were no macroscopic changes due to treatment at 6 or 74 mg/kg/day.

Amongst rats receiving 900 mg/kg/day splenic changes involving haemosiderosis, extramedullary haemopoiesis and more prominent germinal centres in the white pulp were observed. Also at 900 mg/kg/day, 5/5 males showed moderate testicular atrophy. Treatment related changes observed at 74 mg/kg/day were confined to minimal extramedullary haemopoiesis.

Under the conditions of this study the NOAEL of the test material was determined to be 74 mg/kg/day. Changes observed at this dose were considered to be of minor toxicological importance.