Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Aug 1982 to 24 Sep 1982 (Experiment A), 18 Jul 1983 to 19 Aug 1983 (Experiment B)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981 and 2001

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Food and Drug Administration. Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use

Version / remarks:

1966

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Committee on Safety of Medicines. Guidelines on Reproduction Studies for Guidance of Applicants for Product Licences and Clinical Trial Certificates, June.

Version / remarks:

1974

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

EC Number:

276-696-7

EC Name:

Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

Cas Number:

72490-01-8

Molecular formula:

C17 H19 N O4

IUPAC Name:

ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

Test animals

Species:

rabbit

Strain:

other: Swiss Hare

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: Experiment A, 2440-3500 g and experiment B, 2250-3530 g
- Housing: The animals were individually housed in stainless steel cages
- Diet: Diet cubes ad libitum
- Water: Tap water ad libitum
- Acclimation period: Minimum of 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18
- Humidity (%): 50
- Air changes: Fully air- conditioned
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 23 Aug 1982 to 24 Sep 1982 (Experiment A), 18 Jul 1983 to 19 Aug 1983 (Experiment B)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: SSV (4% carboxymethylcellulose, 0.9% NaCl, 0.5% benzylalcohol, 0.4% Tween 80 in water)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was formulated freshly each week in the vehicle. High shear mixing was used during formulation and the suspension was kept homogeneous during dosing by the use of a magneto stirrer

VEHICLE
- Amount of vehicle: 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1:1
- Proof of pregnancy: sperm in vaginal smear referred to as day 1 of pregnancy

Duration of treatment / exposure:

From day 7 to 19 inclusive of gestation

Frequency of treatment:

Once daily

Duration of test:

Until day 30 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Experiment A, group II

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Experiment A, group III

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Experiment A, group IV

Doses / concentrations 4

Dose / conc.:

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

Remarks:

Experiment B, group II

No. of animals per sex per dose:

Experiment A: 20 mated females
Experiment B: 35 mated females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a range finding study for maternal sensitivity and embryotoxicity, the substance doses of 100 and 300 mg/kg bw/day were administered to pregnant rabbits. Based on these results, dosages of 30, 100 and 300 mg/kg bw/day were selected for the principle study
- Foetuses from a necropsy subgroup were removed by ovariohysterectomy one day before parturition and processed for skeletal and visceral examinations. Additionally, dams from a rearing subgroup were allowed to deliver naturally and to raise their young until weaning. All females with litters were necropsied after the 23rd day of lactation and the uteri were examined for implantation sites. The offspring was examined externally.
- Accurate interpretation of the findings from the principle study was increasingly complicated by the appearance of some malformations mainly in the 300 mg/kg dose group which rendered inconclusive results. Therefore, a supplementary study was conducted with increased numbers of females using a dose of 200 mg/kg in an attempt to reproduce the same effects and to establish the relevance of the malformations that arose in the 300 mg/kg group. The same treatment schedule was used as in the principle study and included a separate control group.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: Daily, all changes in behaviour, general condition, signs of pharmacological action, etc., rabbits which during the experiment were autopsied

BODY WEIGHT:
- Time schedule for examinations: On day of gestation 1, 7, 20 and 30

Ovaries and uterine content:

The ovaries and uterine content was examined after termination:
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

Fetal examinations:

- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, all per litter
- Skeletal examinations: Yes, all per litter
- All the young were tested for their 24h viability in an incubator at 34 °C

Statistics:

1. Descriptive statistics:
- In the tables for each group, median and interquartile- range are indicated. The median has the property, that 50 % of all values in the group are smaller and 50 % are larger than the median. The interquartile range contains 50 % of the values in the group, excluding the 25 % smallest and the 25 % largest values.
Exp. A: Statistical evaluations employed the T-TEST for independent samples and/or the CHI—SQUARE—TEST. The data presented in the tables are calculated mean values and standard deviations.
2. Significance tests:
- Trend set: All groups together are tested by a simultaneous test against trend (Jonckheere-test) or Armitage-test as indicated in the table. In case of a significant result, the correspondent interpretation (‘’*’’ for 5 % > p ≥ 1 % and ‘’**’’ for p < 1 % is attached to the highest dosage group. The test is then repeated without the already "significant" highest dosage group and again interpreted as before.
- Simultaneous test without ordered alternative: In the case of a non-significant result of the trend test, the same (remaining) groups are tested by a simultaneous test without trend alternative (Kruskal-Wallis-test and Chi^2-test as indicated in the table). If this test again is not significant (p= 1%) the test procedure ends: all remaining groups are not significantly different from control. P is chosen as 1% for this test to keep the total error probability near 5%
- Two sample tests for each dosage group against control: In the case of a significant result for test b), all dosages groups are separately tested against control by means of a two sample test (U-test or Fisher-test as indicated) the interpretations mean: ‘’#’’: 5% > p ≥ 1% and ‘’##’’: p<1%
3. Experimental unit: The dam is considered the independent experimental unit within all significant tests
4. Relative numbers: (relative number = absolute number/number of implantations)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two dams at 100 mg/kg bw/d died on day 12 and 19 (cause of death unknown).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain was reduced in dams at 300 mg/kg bw/d during the treatment period (80% of control) and at 200 mg/kg bw/d from the treatment period till necropsy (76-90% of control).

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

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:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

2

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal examination of the neonates by radiography and/or Alizarin Red staining revealed no compound- related effects on any of the measured ossification parameters and all findings from the treatment group of both the principle and supplementary studies compared favorably to the respective controls.

Visceral malformations:

no effects observed

Description (incidence and severity):

The examination for soft tissue abnormalities revealed no indication of any drug-related deviation in any dose group

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

In the principle study (A) macroscopic examination of the foetuses at necropsy from the control group revealed one foetus with a hypoplastic tail. A dose of 30 mg/kg revealed one foetus with a hypoplastic tail and two additional litter mates with tails missing. From a dose of 100 mg/kg there were two foetuses with missing tails, one foetus with an open eye and one foetus with spina bifida. The malformations at the highest dose of 300 mg/kg included two cases of spina bifida with hypoplastic tails, one single case from both spina bifida and open eyes, a single foetal resorption with omphalocele and two cases of hypoplastic tail. From the supplementary study (B) with 200 mg/kg the only observed malformation was localized in a foetal resorption which had open eyes and a reduction malformation of the right arm. Contrary to results in study A, the control group of study B contained malformations in which the type and incidence is
analogous to those found in the 300 mg/kg dose group. These included two foetuses with ectopy; one of which had a missing tail, one foetus with an eye open, a foetus with omphalocele and a foetus with hypoplastic tail. Although the malformations arising from study. A were considered as being questionably compound-related, further investigation has undoubtedly shown that an association with the test substance can be excluded. That the observed malformations in study A are not compound- related is mainly the result of substantiating evidence collected from the supplementary study (B). It was assumed that a reproducible pattern of the same types of malformations would have appeared in the 200 mg/kg if those seen in the 300 mg/kg group were actually relevant. But neither pattern nor associated malformations Were established and no relationship exist in the results between the two dose groups. Furthermore, though the incidence of malformations of increased in the 300 mg/kg dose group the same type of malformations are often observed among historical controls but in particular among the controls in the supplementary study. In addition there were also no malformations noted among the foetuses in the preliminary study at doses of 100 and 300 mg/kg. Therefore, in view of the various supporting
data, all of the observed malformations originating in the principle study are considered only to be arbitrary findings

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table 1. Results experiment A

Dose (mg/kg bw/day)	0	30	100	300	dr
Maternal effects Mortality (n=20) Clinical signs Pregnant animals Body weight gain Food consumption Uterus weight Pathology macroscopy	0   18	0            2 No treatment-related findings 20 17   Not performed Not determined   Not performed		0   20   dc
Litter response
Number of dams	18	20	17	20
examined
Corpora lutea/dam		No treatment-related findings
Total number of resorptions/dam	2.2	2.1	1.2	2.8
Number of resorptions/group
- embryonic	38	41	14	50
- foetal	1	2	6	5
Dams with live foetuses	17	17	17	18
Live foetuses/dam	6.8	5.0	6.4	6.3
Dead foetuses	0	0	0	0
Foetal weight	41.8	44.3	44.6	43.6
Post-implantation loss/dam^1	2.1	2.2	1.2	2.7

Dose (mg/kg bw/day)	0	30	100	300	dr
Sex ratio	No treatment-related findings
Mean crown-rump length	No treatment-related findings
Survival rate 24 h	No treatment-related findings
Examination of the foetuses	122   0   1 (0.8%)	101 109   0 1 (0.9%) 1 0 (1.0%) No treatment-related findings No treatment-related findings		126   3 (2.4%) 4 (3.2%)
Total no foetuses
External observations - spina bifida, sacral region - hypoplastic tail
Skeletal findings
Visceral findings

dr: dose related

dc/ic: statistically significantly decreased/increased compared to the controls

d/i: decreased/increased, but not statistically significantly compared to the controls

a/r: absolute/relative organ weight

1: as calculated by the reviewer; no statistical analysis performed

Table 2. Results experiment B

Dose (mg/kg bw/day)	0		200		dr
Maternal effects Mortality Clinical signs Pregnant animals Body weight gain Food consumption Uterus weight Pathology macroscopy	35	None  No treatment-related findings   Not performed Not determined   Not performed		33   d
Litter response
Number of dams examined	35			33
Corpora lutea/dam		No treatment-related findings
Total number of resorptions/dam	1.0			1.0

Dose (mg/kg bw/day)	0		200		dr
Number of resorptions/group - embryonic - foetal	34 11		28 3
Dams with live foetuses	35		33
Live foetuses/dam	7.0		8.0
Foetal weight	42.4		41.6
Post-implantation loss/dam^1	2.0		1.0
Sex ratio	No treatment-related findings
Mean crown-rump length	No treatment-related findings
Survival rate 24 h	No treatment-related findings
Examination of the foetuses	349   0   1 (0.3%)	No treatment-related findings No treatment-related findings		234   0   0
Total no foetuses
External observations - spina bifida, sacralregion - hypoplastic tail
Skeletal findings
Visceral findings

Table 3. Historical control data from Hoffmann LaRoche, between 1977 and 1988

	Control Gronp		Foetuses with Finding
	N of Litters	N of foetuses	hypoplasfictail	missing tail	open eyes	spina bifida	ompha /ocoele
TOTAL	539	3656	3	2	2	2	2
% of litters affected (range)			0-13.3	0-6.7	0-5.3	0-5.6	0-6.7
% of fetuses affected (range)			0-2.3	0-1.1	0-1.0	0-0.8	0-1.1

Applicant's summary and conclusion

Conclusions:

Based on decreased body weight, the NOAEL for maternal toxicity was set at 100 mg/kg bw/day. Based on no effects on foetal development the NOAEL for developmental toxicity was set at ≥ 300 mg/kg bw/day.

Executive summary:

The test substance was tested for embryotoxic and teratogenic action in 20 mated (principle study) and 35 (supplementary study) female Swiss hare rabbits according to OECD TG 414 and GLP principles. In the principle study, doses of 30, 100 and 300 mg/kg bw/day were administered by oral gavage to pregnant animals from day 7 to 19 inclusive of gestation. A control group received the vehicle (SSV, 4% carboxymethylcellulose, 0.9% NaCl, 0.5% benzylalcohol, 0.4% Tween 80 in water) for the same treatment period. All females were sacrificed at day 30 of gestation. Foetuses were removed by ovariohysterectomy tested for viability (24 h) and examined for macroscopic, skeletal and soft tissue anomalies. Accurate interpretation of the findings of the principle study was increasingly complicated by the appearance of some malformations in the 300 mg/kg dose group which rendered inconclusive results. Therefore, a supplementary study was conducted with increased number of females using a dose of 200 mg/kg bw/day in an attempt to reproduce the same effects and to establish the relevance of the malformations that occurred in the 300 mg/kg group. The same treatment schedule was used as in the principle study and included a separate control group. Throughout the summary the principle and supplementary study will be referred as A and B.

Results showed a moderately reduced body weight development during the treatment period among females of the 200 and 300 mg/kg dose groups, but not a highly significant effect. There were no detectable effects on the measured reproductive parameters, on the course and outcome of pregnancy nor on the 24 hour survivability of the neonates in either study. In the principle study (A) beginning with a dose of 100 mg/kg, some morphological malformations appeared mainly as open eyes, spina bifida and/or short or missing tail. The incidence was low and compared favourably to the frequency of these malformations in observed historical control data of this rabbit strain. In spite of no effects seen in the preliminary study at a dose of 300 mg/kg, malformations did appear at this dose level in the principle study. The malformations were similar to those observed in the 100 mg/kg group but the incidence was somewhat higher than recognized in historical controls thus, complicating an accurate interpretation of the results. In the supplementary study (B) using a dose of 200 mg/kg, it was assumed that a pattern of the same types of malformations would appear if the effects seen in the 300 mg/kg group were relevant. However, the results from this study show that no pattern or associated malformation was established between the two doses and only one malformation was present and localized in a foetal resorption. Furthermore, in contrast to the single malformation from the control group of the principle study (A), the malformations seen in the control group of the study supplementary study (B) appeared more frequently and were analogous in both type and incidence to the malformations seen in the 300 mg/kg dose group. Therefore, in view of all substantiating evidence with particular emphasis on the high incidence of malformations seen in the controls of the supplementary study, all malformations seen in the principle study (A) of the 300 mg/kg group are considered to be arbitrary findings and not test substance-related. Examination of the foetuses for skeletal and soft tissue anomalies did not reveal any effects considered to be test substance-related from any dose group.

In conclusion, based on decreased body weight, the NOAEL for maternal toxicity was set at 100 mg/kg bw/day. Based on no effects on foetal development the NOAEL for developmental toxicity was set at ≥ 300 mg/kg bw/day.