Diethyl phthalate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Experimental result published in peer reviewed journal. Some details on methods / results not available

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

DEP was administered to time-mated rats in the diet during the period of organogenesis and maternal toxicity and effects on embryo-foetal viability, growth and morphogenesis assessed.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diethyl phthalate

EC Number:

201-550-6

EC Name:

Diethyl phthalate

Cas Number:

84-66-2

Molecular formula:

C12H14O4

IUPAC Name:

.

Details on test material:

- Name of test material (as cited in study report): Dietyl phthalate (DEP)
- Analytical purity: >99%
:

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc Raleigh NC USA
- Weight at study initiation: females 0gd 207-273 g
- Fasting period before study: No
- Housing: individually in solid bottom polycarbonate cages with stainless steel wire lids (Laboratory products Rochelle Park NJ USA
- Diet (e.g. ad libitum): yes, ground Purina Certified Chow #5002
- Water (e.g. ad libitum): yes, deionised water
- Acclimation period: 7d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 degrees C
- Humidity (%): 48%
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: control feed

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food): mixed with feed in a Patterson Kelley Liquids -Solids twin shell blender
- Storage temperature of food: refridgerated in containers protected from light

VEHICLE
- Justification for use and choice of vehicle (if other than water): control feed, DEP was administered in the diet
- Concentration in vehicle: 0, 0.25, 2.5 & 5.0%
- Lot/batch no. (if required): not reported but fed ground Purina Certified Chow #5002

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diets were analysed by HPLC before & after use. Analysis confirmed that the diets contained 89-110% of the nominal concentrations.

Details on mating procedure:

- Impregnation procedure: [artificial insemination / purchased timed pregnant / cohoused]: Time mated rats
- If cohoused: bred in pairs
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight until sperm detected in the vagina
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: sperm in vaginal smear = gd0

Duration of treatment / exposure:

From morning of gd6 to morning of gd15.

Frequency of treatment:

Continuously in the diet

Duration of test:

Until GD 20

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 0.25, 2.5 & 5.0%
Basis:
nominal in diet

No. of animals per sex per dose:

25-32 gravid females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: To include and exceed the range of doses used in earlier study (ip study in 5 animals/group by Singh et al 1972 used 0.57, 1.13 & 1.89 mg/kg during gestation period reported to cause effects onfoetal growth & development including an increase in the incidence of skeletal defects)
- Rationale for animal assignment (if not random): assignment was by stratified randomisation so that body weights were simalar in the groups within each of 2 replicates
- Other: range of 3-4 consecutive breeding days in each replicate. The replicates were 26 days apart

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: d: 0, 3, 6, 9, 12, 15, 18, & 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes over 3 day periods from gd6-20 ie gd6-9, 9-12, 12-15, 15-18, 18-20

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: gd6-9, 9-12, 12-15, 15-18, 18-20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Not clearly stated

OTHER: Body weight, & liver, kidney and intact uterus weights were recorded. Uteri with no visible implantation sites were stained with 10% amonium sulfide to detect implantation sites from early resorptions.

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

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes

Statistics:

SAS (GLM procedures)software was used for the analysis of variance of maternal & foetal parameters. When ANOVA revealed a significant P<0.05) dose effect Williams & Dunnetts tests were used to compare each treatment group with the concurrent control group. One-tailed were used in the pair wise comparisons except those of body weigt & organ weight. For non significant(p>0.05 dose X replicate effects on foetal parameters data were pooled accross the replicates and non parametric tests applied. When the X2 test indicated significant differences among all groups a one tailed Fisher's exact probability test was used to compare individual treatment groups with the concurrent control group. The alpha level for ball groupwise and pairwise comparisons was 0.05.

Indices:

Not reported

Historical control data:

Not reported

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: Body weight & body weight gain were reduced in dams given 2.5 and 5% DEP in the diet from on and from 9 respectively. See table below. Food & water consumption was also reduced in these groups during gd 6-9 & 9-12 respectively and increased during in the

Details on maternal toxic effects:
See table below

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

0.25 other: %

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

2.5 other: %

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: A signicantly increased incidence of variations as a result of the increase in rudimentary extra lumbar ribs in the 5% dose group when compared with the controls. See tables below.

Details on embryotoxic / teratogenic effects:
See tables below

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Maternal toxicity in CD rats exposed to diethyl phthalate in feed on gestational days 6 to 15

	Diethyl phthalate (% in feed)
	0	0.25	2.5	5.0
Subjects (dams)
Total treated	31	32	32	32
Removed	0	1^1	0	0
No. (%) pregnant	27(87)	29(94)	30(94)	32(100)
Chemical exposure (g/kg/day)^2
gd 6 to 9	0	0.21	1.65	1.95
gd 9 to 12	0	0.20	1.97	3.43
gd 12 to 16	0	0.19	2.06	4.39
gd 6 to 16	0	0.20	1.91	3.21
Maternal wt gain (g)^3
Gestation period	152.8±4.0^†	169.2±3.4^*	154.8±3.2	145.8±3.9
Treatment period	52.8±2.0^†	61.4±1.4^*	52.6±1.6	30.6±3.2^*
Corrected wt gain^4	66.4±3.9^†	78.4±2.4^*	67.1±2.7	57.0±3.2^*
Gravid uterine wt (g)^3	86.0±3.3	90.9±2.6	87.7±2.2	88.7±1.9
Relative food consumption (g/kg/day)^2
gd 6 to 9	82.3±1.5^†	82.3±1.2	65.9±3.7^*	39.0±6.1^*
gd 9 to 12	78.8±1.1^†	79.7±2.9	78.6±2.3	68.5±3.9^*
gd 12 to 16	76.7±1.2^†	76.7±1.8	82.5±1.2^*	87.7±2.0^*
gd 16 to 18	77.9±1.3^†	79.3±1.3	80.2±1.6	92.2±2.4^*
gd 18 to 20	73.2±2.4^†	73.2±1.6	76.3±2.0	83.0±3.2^*
gd 0 to 20	77.9±0.8	77.2±1.0	77.2±0.9	75.6±1.1
Relative water consumption (g/kg/day)^2
gd 6 to 9	143.3±4.7^†	133.5±5.0	121.9±4.1^*	119.8±4.9^*
gd 9 to 12	143.3±6.0	141.2±4.6	140.8±4.2	154.4±5.6
gd 12 to 16	154.6±14.8^†	135.0±3.4	150.7±10.8	180.6±8.3
gd 16 to 18	138.8±9.2^†	138.2±4.8	148.4±4.1	180.6±5.4^*
gd 18 to 20	134.6±6.4^†	126.3±2.4	135.3±3.0	158.0±5.3^*
gd 0 to 20	142.1±5.7^†	133.6±3.4	137.6±3.6	148.9±3.7

_{1 Dams were removed because of insufficient food or water or for being outside of the weight range}

_{2 Food consumption during treatment (g/day) x % chemical x 1000 mg/g / average body wt during treatment (kg)}

_{3 Includes all dams pregnant at sacrifice, mean±SB}

_{4 Weight gain during gestation minus gravid uterine weight}

_{* Dunnett’s test or Williams’ test, P<0.05}             

_{†Linear trend test, P<0.05}

Developmental toxicity in CD rats following maternal exposure to diethyl phthalate in feed on gestational days 6 to 15

	Diethyl phthalate (% feed)
	0	0.25	2.5	5.0
All litters^1	27	29	30	32
No. implant sites/litter	15.2±0.5	15.8±0.4	15.0±0.4	15.5±0.3
% resorptions/litter	3.8±1.4	3.9±0.9	4.1±1.4	3.1±0.7
% litters with resorptions	25.9	48.3	26.7	40.6
Live litters^4	27	29	30	32
No. live foetuses/litter	14.6±0.5	15.2±0.5	14.4±0.4	15.0±0.3
% male foetuses/litter	50.4±2.8	48.3±2.8	48.7±2.8	50.2±2.5
Mean body wt. (g)/litter	3.7±0.2	3.9±0.1	3.9±0.1	3.8±0.0
% foetuses malformed/litter	0.7±0.5	0.4±0.3	0.5±0.3	1.3±0.6
% litters with malformed foetuses	7.4	6.9	6.7	15.6
% foetuses with variations/litter	10.6±3.1^t	8.4±2.2	12.7±2.8	23.5±4.1^*
% litters with variations	51.9^+	44.8	66.7	84.4^†
% foetuses with extra rib/litter	8.8±3.0^†	7.1±2.0	10.6±2.9	21.0±4.1^*
% litters with extra rib	44.4^+	39.3	46.7	74.2^†

_{1 Includes all dams pregnant at sacrifice litter size = no. implantation sites per dam: mean±SE; 2 One dam had a litter that contained only one dead foetus}

_{3 One dam had 100 resorptions; 4 Includes only dams with live foetuses litter = no. live foetuses per dam: mean±SE}

_{†Linear trend test, P<0.05; * Dunnett’s test or Williams’ test, P<0.05; +x2 test, P<0.05;tFisher’s exact test, P<0.05}

 

Morphologic defects in CD rat fetuses following maternal exposure to diethyl phthalate in feed on gestational days 6 to 15

	Diethyl phthalate (% of feed)
	0	0.25	2.5	5.0
All malformations
Litters with malformations/total litters^3	2/27	2/29	2/30	5/32
Malformed foetuses/total examined^4	3/394	2/442	2/431	6/479
External malformations
No. litters with defects^5	0	1	0	2
No. foetuses with defects^6	0	1	0	2
Exencephaly	0	1	0	1
Spina bifida	0	1	0	1
Microphthalmia	0	0	0	1
Visceral malformations
No. litters with defects^5	2	0	0	3
No. foetuses with defects^6	3	0	0	4
Hydroureter	3	0	0	2
Hydronephrosis	0	0	0	2
Enlarged lateral ventricle of brain	0	0	0	1
Extra renal vessel	0	0	0	1
Skeletal malformations
No. litters with defects^5	0	2	2	0
No. foetuses with defects^6	0	2	2	0
Fused rib cartilage	0	1	0	0
Short rib XIII	0	1	0	0
Rib cartilage not attached to sternum	0	1	2	0
Variations
No. litters with defects^5	14	13	20	27
No. foetuses with defects^6	42	38	54	117
Hematoma	3	2	4	2
Open eye	0	0	0	1
Visceral variations
Agenesis of the innominate artery	0	0	0	2
Pale area on liver	0	0	0	1
Distended ureter	3	3	1	3
Skeletal variations
Misaligned sternebra	1	0	2	0
Wavy rib		1	1	2
Extra rib (rudimentary)	32	32	44	100
Extra rib (full)	3	0	1	7
Incomplete ossification with cartilage, frontals and/or parietals (midline)	0	0	1	3
Normal cartilage, bipartite ossification centre of vertebral centrum	1	3	0	3
Dumbbell cartilage, bipartite ossification centre of vertebral centrum	0	0	0	0

_{1 Statistical evaluation of these data is reported in Table 2; 2 Foetuses may have more than one defect; 3 Includes only litters with live foetuses}

_{4 Only live foetuses were examined for defect;5Litters with one or more defective foetuses; 6 Foetuses with one or more defects}

 

Applicant's summary and conclusion

Conclusions:

Under the conditions in which this study was performed the NOAELs for maternal & developmental toxicity were 0.25% & 2.5% DEP in the diet due to the reduced maternal body weight at the 2.5 (transient) & 5% dietary concentrations & the increase in the incidence of supernumery ribs in the foetues exposed to 5% DEP in the diet.

Executive summary:

Developmental toxicity has been investigated in the rat using methods similar to those described by OECD test guidelines. Diethyl phthalate was administered in the diet during gestation days 6 -15. The NOAELs for maternal & developmental toxicity were 0.25% (equivalent to 200 mg/kg) and 2.5% (equivalent to 1910 mg/kg) due to the reduced maternal body weight at the 2.5 (transient) & 5% dietary concentrations and an increase in the incidence of supernumery ribs in the foetues exposed to 5% DEP in the diet.