Ethyl 4-dimethylaminobenzoate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 May 1998 to 19 October 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl 4-(dimethylamino)benzoate

EC Number:

244-289-3

EC Name:

2-ethylhexyl 4-(dimethylamino)benzoate

Cas Number:

21245-02-3

Molecular formula:

C17H27NO2

IUPAC Name:

2-ethylhexyl 4-(dimethylamino)benzoate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

HsdCpb:WU

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann, Versuchstierzucht, in 33178 Borchen/Paderborn
- Age at study initiation: 11 weeks old at start of mating
- Weight at study initiation: 191 g (min. and max. values 173 and 208 g)
- Housing: The rats were housed in cages in air conditioned rooms. Softwood granulate specially manufactured for housing of animals served as the bedding material.
- Diet: Standard diet ad libitum
- Water: Drinking water ad libitum
- Acclimation period: 6 days before the start of mating

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 22 °C
- Humidity: 53 to 75 % (relative)
- Air changes: Not specified; rooms were air conditioned
- Photoperiod: 12 hours of light per day (6:00 a.m. to 6:00 p.m.).

IN-LIFE DATES
From: 16 June 1998
To: 15 July 1998

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Miglyol 812 neutral oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The preparations of the test material were produced once a week at the testing facility central dispensary. The preparations were provided in amber glass bottles. The preparations were stored at room temperature in the dark. After the contents of the bottles had been mixed thoroughly, the daily rations were filled into brown flasks provided with a magnetic rod. For administration, the flasks were put on a stir bar and stirred continuously.
- Dose volume: 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

According to stability tests, the test material is at least stable for 14 days in the preparation used.
The concentration and homogeneity of test material in the preparations administered were determined once during the study. The results can be seen in table 1.
During preparation of the samples for analysis, homogenisation was not performed correctly. This led to the insufficient concentrations in the analysis. For this reason the values are not considered to be valid. It is to be assumed that the animals received the required amounts of the test material.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1 M: 4 F
- Length of cohabitation: Overnight. Groups of 4 sexually mature, virgin females were left overnight with one stock stud.
- Proof of pregnancy: On the following morning co-housing, vaginal smears were taken to check for the presence of sperm. The day when sperm was detected was defined as gestation day 0 (GD 0)

Duration of treatment / exposure:

14 consecutive days from GD 6 to 19

Frequency of treatment:

Once daily

Duration of test:

20 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a dose-finding study 5 rats each were treated with oral doses of 100, 300 and 1000 mg /kg of test material from gestation days 6 to 19. At the dose level of 100 mg/kg a slight maternal toxicity could not be excluded. 300 mg had a weak maternotoxic effect: rooting in the bedding and a lower terminal body weight were observed. 1000 mg/kg had a clear maternotoxic effect. In addition to the findings observed at the low and mid doses, reduced food consumption, lower body weight gain and lighter foetuses were observed at this high dose. Based on these results the dose of 40 mg/kg was chosen as the low dose for the main study, at which no toxic effect was expected to occur. 1000 mg/kg was selected as the high dose, as this dose had led to a clear maternotoxic effect, which was expected to occur at the high dose level in the present study as well. The mid dose of 200 mg/kg is the geometric mean of the low and high doses.
- Animal assignment: Using the randomisation generator VS APL 4.0 on an IBM computer.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked included: behaviour and appearance

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 6 and then daily until GD 20.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: GD 6, 10, 15 and 20.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Every 3 days

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # GD 20
On GD 20 all rats were killed with halothane in random sequence. Their abdominal cavities were opened and their ovaries removed together with the uteri. The bodies without uteri and foetuses were weighed and recorded as terminal body weights.

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
The uteri (containing the foetuses) were weighed. The number of corpora lutea in both ovaries was determined. After opening the uteri the numbers of live and dead foetuses and the numbers of resorptions, as well as their distribution to the left and right uterine horns, were recorded.

The following types of resorption were differentiated:
- Complete resorptions: Resorptions that had occurred shortly after implantation and were detected after staining of the uteri with ammonium sulphide.
- Early resorptions: Diameter of up to 0.5 cm. No macroscopic differentiation between foetal and placental residues possible.
- Late resorptions: Diameter of usually more than 0.5 cm, clear macroscopic differentiation between foetal and placental residues possible.
The total number of implantations was determined by staining the uteri with ammonium sulphide. Females without implantation sites were recorded as non-pregnant.

Blood sampling:

no

Fetal examinations:

- External examinations: Yes: all per litter were examined macroscopically for abnormal deviations and weighed. Their sex was determined on the basis of the anogenital distance and additionally by inspection of the gonads.
- Soft tissue examinations: Yes: foetuses from a randomised half of females were scheduled for transverse section. The foetuses were fixed in formalin and dissected transversely (method after Wilson, 1964) and inspected for organ malformations (modified method after Barrow and Taylor 1967).
- Skeletal examinations: Yes: foetuses from the other half of the females were eviscerated, fixed in alcohol and treated according to the double staining method of Whitaker and Dix (1979); i.e. the soft parts were cleared with potassium hydroxide solution, the skeletons were stained with Alizarin Red S, and the cartilage with Alcian Blue. The foetuses were then examined for skeletal abnormalities and malformations.

Statistics:

Body weight and body weight gain of females, terminal body weight (body weight without uteri and foetuses, Uterus weights (containing the foetuses), food and water consumption and foetus weights were evaluated using a two-sided Dunnett test.
Corpora lutea, implantations were evaluated using a two-sided Fisher-Pitman Permutation test. Live foetuses, dead foetuses, resorptions, malformed foetuses, % live foetuses (related to implantations) and % post-implantation loss (resorptions and dead foetuses related to implantations) were evaluated using a one-sided Fisher-Pitman Permutation test.

Historical control data:

no

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

As a reaction to treatment, 12/25 rats of the mid dose group and 22/25 rats of the high dose group showed salivation after dosing for about 15 minutes on different days. Rooting in the bedding material for about 15 minutes after dosing on different days was seen in all rats of the mid and in all rats of the high dose group.
Furthermore, hair loss was observed in two rats of the control, low and high dose groups, and in 4 rats of the mid dose group. In the mid and high dose groups one rat each had a scabby skin wound. In the mid dose group one rat had a blood crusted eye. These single findings are not regarded as being treatment related.

Mortality:

no mortality observed

Description (incidence):

None of the animals died.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight development in the low dose group corresponded to that in the control, but it was very slightly (not significantly) lower in the mid dose group.
The body weight in the high dose group was significantly reduced from GD7 until necropsy on GD 20 (at GD20 -10% when compared to the controls) associated with lower boduweight during during all the treatment period (at GD20: -28% when compared to the controls).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption in the low and mid dose groups did not differ significantly from the control, but in the high dose group it was found to be significantly reduced after start of dosing until necropsy.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

The water consumption in the low dose group did not differ significantly from the control. It was slightly (significant only for GD 12 to 15 and 18 to 20) increased in the mid dose group and significantly increased in the high dose group from GD 6 until the animals were killed.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

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):

The terminal body weight in the low and mid dose groups did not differ significantly from the control, but was significantly lower in the high dose group.

The absolute and relative uterus weights in the low and mid dose groups were similar to the control, but were lower (absolute weight significantly) in the high dose group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no abnormal findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The frequency of the numbers of implantations in the low, mid and high dose groups corresponded to that in the control.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The resorption rates in the low, mid and high dose groups did not differ significantly from that in the control.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The resorption rates in the low, mid and high dose groups did not differ significantly from that in the control.

Dead fetuses:

no effects observed

Description (incidence and severity):

The frequencies of live foetuses in the low, mid and high dose groups were not statistically different form the control. One dead foetus was seen each in the control and low dose group.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The numbers pregnant per group were 24, 23, 23 and 21 per group for the control, low, mid and high dose groups, respectively.

Other effects:

no effects observed

Description (incidence and severity):

The frequency of the numbers of corpora lutea in the low, mid and high dose groups corresponded to that in the control.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal toxicity

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

2

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Remarks on result:

other: no test item-related effect observed at this dose

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The weights of the male and female foetuses in the low dose group were similar to the control. The foetuses in the mid dose group were slightly lighter when compared with controls (-3/5%), but the weights did not differ significantly from the control. In the high dose group, the foetuses were significantly lighter in comparison to the controls (-25% in males and females).

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The frequencies of live foetuses in the low, mid and high dose groups were not statistically different form the control. One dead foetus was seen each in the control and low dose group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The ratio of male to female foetuses in the low, mid and high dose groups did not differ significantly from the control.

Changes in litter size and weights:

not examined

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

The ossification status of the foetuses correlated with their body weight. Corresponding to the low body weight, the sternum, coccygeal vertebrae, head bones and extremities in the high dose group were less ossified. A trend to a slightly lower ossification was also seen in the mid dose group. A larger number of kinked ribs was observed in the high dose group as well.

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

No teratogenic effect was found.

Effect levels (fetuses)

1

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no test item-related effect observed at this dose

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

200 mg/kg bw/day

Treatment related:

yes

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the no observed adverse effect level (NOAEL) for maternal toxicity and for developmental toxicity was 200 mg/kg/day. At 1000 mg/kg/day, a lower maternal bodyweight was associated to the lower foetuses bodyweight and retarded skeletal ossification. No teratogenic effect was found in this study.

Executive summary:

The potential for the test material to cause developmental toxicity via the oral route in the rat was investigated in a study conducted in accordance with the standardised guideline OECD 414 under GLP conditions.

The test material was administered by gavage to pregnant Wistar rats from gestation days (GD) 6 to 19 at daily doses of 40, 200, and 1000 mg/Kg. The control rats in group 1 were treated with the vehicle Miglyol 812 neutral oil. The foetuses were delivered by caesarean section on GD 20 and were examined for macroscopic malformations. Half of them were examined for skeletal malformations and half for organ malformations. The terminal body weights of the dams were recorded.

Rooting in the bedding material was seen in all rats in the mid and high dose groups. alivation was seen in half the animals of the mid and in most animals of the high dose group. None of the animals died. Twenty one to 24 rats out of 25 per group proved to be pregnant.

The body weight gain in the mid dose group was very slightly and in the high dose group clearly reduced. The food consumption was reduced in the high dose group. The water consumption in the mid and high dose groups was dose dependently increased. The terminal body weight and the uterus weight were decreased in the high dose group. The numbers of corpora lutea, implantations and live foetuses were not affected. The number of dead foetuses, complete, early and late resorptions were not increased. The sex distribution was not affected in any of the groups. The foetuses in the mid dose group were slightly and in the high dose group clearly lighter than those of the controls.

Despite a trend towards retarded ossification in the mid dose group and a clearly retarded ossification in the high dose group, corresponding to the low foetus weights, the skeletal examinations gave no indication of malformations attributable to the treatment with the test material. The frequency of all malformations was in a usual range.

In conclusion, the dose of 40 mg/kg of test material was systemically tolerated by the rats. The dose of 200 mg/kg led to slight maternotoxic effects, such as rooting in the bedding material, salivation, slightly reduced body weight gain and higher water consumption. The foetuses were slightly lighter and there was a trend towards lower ossification. The dose of 1000 mg/kg led to clear maternotoxic effects, expressed by salivation and rooting in the bedding material, reduced body weight gain and reduced food consumption. The foetuses were clearly lighter and the ossification was retarded at 1000 mg/kg/day.

Under the conditions of this study, the no observed adverse effect level (NOAEL) for maternal toxicity and for developmental toxicity was 200 mg/kg/day. At 1000 mg/kg/day, a lower maternal bodyweight was associated to the lower foetuses bodyweight and retarded skeletal ossification. No teratogenic effect was found in this study.