Bis(2-propylheptyl) phthalate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-propylheptyl) phthalate

EC Number:

258-469-4

EC Name:

Bis(2-propylheptyl) phthalate

Cas Number:

53306-54-0

Molecular formula:

C28H46O4

IUPAC Name:

1,2-bis(2-propylheptyl) benzene-1,2-dicarboxylate

Test material form:

other: clear colourless liquid

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Strain: Wistar (CrIGIxBrlHan:WI)
- Age at study initiation: 70 - 84 at delivery
- Weight at study initiation: 146.3 - 188.6 g
- Housing: single, DK III stainless steel wire mesh cages (Becker, Castrop-Rauxel, Germany)
- Diet (e.g. ad libitum): ground Kliba laboratory diet rat/mouse/hamster, Provimi Kliba SA, Kaiseraugts, Switzerland, ad libitum
- Water (e.g. ad libitum): drinking water; ad libitum
- Acclimation period: between the supply on day 0 and the first administration on gestational day 6


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance solutions in olive 011 Ph.Eur./DAB were prepared at the beginning of the administration period and thereafter at intervals, which took into account the analytical results of the stability verification. For the preparation of the solutions, an appropriate amount of the test substance was weighed in a graduated beakers (depending on the dose group), topped up with olive oil Ph.Eur./DAB and subsequently thoroughly mixed using a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0.8, 4, 20%
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in olive oil for a period of at least 7 days at room temperature were carried out by HPLC or GC.

Details on mating procedure:

Mating procedure: the animals were mated by the breeder (time-mated) and supplied on day 0 post coitum
- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

day 6 through day 19 of gestation

Frequency of treatment:

daily

Duration of test:

20 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

40 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: a check was made twice a day on working days or once a day (Saturday, Sunday or on public holidays) (days 0 - 20 p.c.).
- Cage side observations checked in table were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: the animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited (days 0 - 20 p.c.).


BODY WEIGHT: Yes
- Time schedule for examinations: all animais were weighed on days 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 p.c.. The body weight change of the animals was caiculated from these results.


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


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 p.c.
- Organs examined: uterus, ovaries

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
- Other: dead fetuses

Fetal examinations:

- External examinations: Yes:
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

Statistical analysis: Dunnett's test (food consumption, body weight, body weight change, corrected body weight gain, weight of uterus (before opening), weights of fetuses, placentae, corpora lutea, implantations, pre- and post implantation losses, resorptions and live fetuses). KRUSKAL-WALLIS-test (weights of liver, kidneys, spleen). Fisher's exact test (conception rate, mortality of the dams, number of litter with fetal findings). Wilcoxon test (proportion of fetuses with malformations, variations and/or unclassified observation in each litter).

Indices:

- The conception rate (in %) was calculated according to the following formula: number of pregnant animals/number of fertilized animals x 100
- The preimpIantation loss (in %) was calculated according to the following formula: (number of corpora lutea - number of implantations) / number of corpora lutea x 100
- The postimplantation loss (in %) was calculated from the following formula: (number of implantations - number of live fetuses) / number of implantations x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In total 5 high dose dams showed urine-smeared fur occasionally, predominantly on the last days of the treatment period (days 16 - 20 p.c.).
The clinical finding "urine smeared fur" is a sign of discomfort of the rats and is probably substance-induced. There were no abnormal clinical findings in the other dams of this study.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/d: The mean body weights of the high dose rats were statistically significantly below control values between days 19 - 20 p.c.. On day 20 p.c., the mean body weight of these rats was about 6% below the mean value of the concurrent control females. There was a statistically significant body weight less at 1,000 mg/kg bw/d at initiation of treatment (days 6 - 8 p.c.). This is in-line with the reductions in food intake of these dams during this study phase. Thereafter, weight gains of the high dose dams were generally slightly below corresponding control values without attaining statistical significance. If calculated for the entire treatment period (days 6 - 19 p.c.), however, weight gain of the high dose dams was statistically significantly impaired and about 24% below the respective contral value. A reduction of about 18% occurred at 1,000 mg/kg bw/d, if weight gain was calculated for the total study phase (day 0 - 20 p.c.).
The corrected body weight gain (terminal body weight on day 20 p.c. minus weight of the unopened uterus minus body weight on day 6 p.c.) was distinctly and statistically significantly lower at 1,000 mg/kg bw/d (about 30% below the concurrent control
value).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/d: mean food consumption was statistically significantly reduced (up to about 32% below the concurrent control value) on treatment days 6 - 13 p.c.. On the following days of the treatment period, food consumption reached or even exceeded control values. If calculated for the entire treatment phase (days 6 - 19 p.c.), food uptake of the 1,000 mg/kg bw/d dams was still about 11 % below the comparable control value. The transient reductions in food consumption at 1,000 mg/kg bw/d were accompanied by corresponding impairments in body weight gain of these dams at initiation of dosing. Therefore, this finding is considered to be substance-induced.

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

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

UTERUS WEIGHT
1000 mg/kg bw/d: the mean gravid uterus weight of the high dose females, was about 19% below the control value, but the difference did not attain statistical significance due to the high standard deviation. The 3 dams of this group which had no live fetuses at all, but only early resorptions had very low uterus weights, whereas the uterus weights of the other dams of this group remained unaffected.

Gross pathological findings:

no effects observed

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:

not specified

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: the postimplantation lass value in the top dose was statistically significantly increased (23.1%) and distinctly outside the historical control range (mean value: 6.6%; range: 3.7 - 11.3%). The increased embryo-/fetolethality at 1,000 mg/kg was not equally distributed throughout the dams of this dose group, but was primarily induced by 3 high dose dams which had no live fetuses at all, but only very early resorptions. Thus, the 3 affected dams were only pregnant by stain and had a postimplantation lass of 100% each, whereas the postimplantation loss values of the other 1,000 mg/kg dams were generally similar to control values.

Total litter losses by resorption:

not specified

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/d: a statistically significant increase in resorption rate (especially early ones).

Dead fetuses:

not specified

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Changes in number of pregnant:

not specified

Other effects:

no effects observed

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

not specified

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not specified

External malformations:

no effects observed

Description (incidence and severity):

No external malformations or variations were seen in any of the fetuses of test groups, except two unclassified variations which were regarded as isolated and considered to be spontaneous in nature.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Malformations of the skeletons were observed at low incidences in fetuses of the test groups 40 and 1,000 mg/kg bw/d. In total, none out of 115 control fetuses (from 24 litters), 2 out of 105 low dose fetuses (1.9%) in 2 out of 23 litters (8.7%), none out of 114 mid dose fetuses (from 25 litters) and 2 out of 77 high dose fetuses (2.6%) in one of 17 litters (5.9%) showed skeletal malformations. The mean percentages of affected fetuses/litter with skeletal malformations amounted to 0.0, 1.6, 0.0, and 2.4%. The noted skeletal malformations appeared without a clear relation to dosing, without biologically relevant differences between the groups and/or can be found at a comparable frequency in the historical control data
In all groups, signs of skeletal variations with or without involvement of corresponding cartilaginous structures elicited. The mean percentages of affected fetuses/litter with skeletal variations amounted to 96.0, 95.0, 97.4, and 100% at 0; 40; 200 or 1,000 mg/kg bw/d.
However, two skeletal variations, occurred at statistically significantly rates in the high fetuses (1,000 mg/kg bw/d). The increased occurrence of unossified sternebra and supernumerary rib (without cartilage) were clearly above the upper historical-control values. However, the overall rate of skeletal variations at this dose level was not statistically significantly elevated above the concurrent control value (affected fetuses/litter: 96.0, 95.0, 97.4 and 100.0% in all groups).
Also, unclassified cartliage observations, occurred in all groups including the controls. The mean percentages of affected fetuses/litter with these findings amounted to 38.6, 34.6, 47.2, and 63.5%** at 0; 40; 200 or 1,000 mg/kg bw/d (** = p< 0.01). A toxicological relevance for these findings could be excluded.

Visceral malformations:

no effects observed

Description (incidence and severity):

No soft tissue malformations were recorded in any of the fetuses of all groups.
However, soft tissue variations were detected in each group including the control. Uni- or bilateral dilations of the renal pelvis occasionally in association with uni- or bilaterally dilated ureter(s) were found in fetuses of all groups. The mean percentages of affected fetuses/litter with total soft tissue variations were within the historical control range (4.0 - 22.2%) in test groups (8.5% (control), 9.1% (40 mg/kg bw/d) and 14.8% (200 mg/kg bw/d)), but were above the upper historical control value at 1,000 mg/kg bw/d (26.7%; p< 0.05).

No so-called unclassified soft tissue observation (like blood inhibition of kidneys) was recorded in any of the fetuses.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
WEIGHT
The mean fetal body weights in test groups were not influenced by the test substance administration and were very similar to or even identical with concurrent control values.

SEX DISTRIBUTION
The sex distribution of the fetuses in the test groups 1 - 3 was comparable with that of the control fetuses. The differences observed in comparison to the control were without any biological relevance.

EXTERNAL EXAMINATION
No external malformations or variations were seen in any of the fetuses of test groups, except two unclassified variations which were regarded as isolated and considered to be spontaneous in nature.

SOFT TISSUE EXAMINATION
No soft tissue malformations were recorded in any of the fetuses of all groups.
However, soft tissue variations were detected in each group including the control. Uni- or bilateral dilations of the renal pelvis occasionally in association with uni- or bilaterally dilated ureter(s) were found in fetuses of all groups. The mean percentages of affected fetuses/litter with total soft tissue variations were within the historical control range (4.0 - 22.2%) in test groups (8.5% (control), 9.1% (40 mg/kg bw/d) and 14.8% (200 mg/kg bw/d)), but were above the upper historical control value at 1,000 mg/kg bw/d (26.7%; p< 0.05).
No so-called unclassified soft tissue observation (like blood inhibition of kidneys) was recorded in any of the fetuses.

SKELETAL EXAMINATION
Malformations of the skeletons were observed at low incidences in fetuses of the test groups 40 and 1,000 mg/kg bw/d. In total, none out of 115 control fetuses (from 24 litters), 2 out of 105 low dose fetuses (1.9%) in 2 out of 23 litters (8.7%), none out of 114 mid dose fetuses (from 25 litters) and 2 out of 77 high dose fetuses (2.6%) in one of 17 litters (5.9%) showed skeletal malformations. The mean percentages of affected fetuses/litter with skeletal malformations amounted to 0.0, 1.6, 0.0, and 2.4%. The noted skeletal malformations appeared without a clear relation to dosing, without biologically relevant differences between the groups and/or can be found at a comparable frequency in the historical control data
In all groups, signs of skeletal variations with or without involvement of corresponding cartilaginous structures elicited. The mean percentages of affected fetuses/litter with skeletal variations amounted to 96.0, 95.0, 97.4, and 100% at 0; 40; 200 or 1,000 mg/kg bw/d.
However, two skeletal variations, occurred at statistically significantly rates in the high fetuses (1,000 mg/kg bw/d). The increased occurrence of unossified sternebra and supernumerary rib (without cartilage) were clearly above the upper historical-control values. However, the overall rate of skeletal variations at this dose level was not statistically significantly elevated above the concurrent control value (affected fetuses/litter: 96.0, 95.0, 97.4 and 100.0% in all groups).
Also, unclassified cartliage observations, occurred in all groups including the controls. The mean percentages of affected fetuses/litter with these findings amounted to 38.6, 34.6, 47.2, and 63.5%** at 0; 40; 200 or 1,000 mg/kg bw/d (** = p< 0.01). A toxicological relevance tor these findings could be excluded.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: embryotoxicity

2

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fetotoxicity

3

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Maternal examination:

Dose (mg/kg bw/d)	0	40	200	1000
Mated females	25	25	25	25
Pregnant females	24	23	25	20
Mortality of dams	0	0	0	0
Pregnant on C-section	24	23	25	20
Clinical symptoms:	-	-	-	+
Food consumption day 6-19 p.c. g	16.9	17.2	17.6	15.1
Mean body weight day 0 (g)	169.2	170.7	173.2	171.9
Mean body weight day 6 (g)	196.9	197.1	201.7	198.3
Mean body weight day19 (g)	265.2	264.4	271.1	250.0*
Mean body weight gain (days 6-19; (g))	68.4	67.4	69.4	51.7**
Uterus weight (g)	49.4	47	48.7	40.2

+ urine smeared fur

*p<0.05; ** p<0.01 (Dunnett test or Kruskal-Wallis and Wilcoxon)

Caesarian section/fetal examination:

Dose (mg/kg bw/d)	0	40	200	1000
Corpora lutea (mean)	10.1	9.8	10.5	10.6
Implantation sites (mean)	9.6	9	9.4	9.4
Post-implantation loss (mean %)	6.2	4.2	6.4	23.1**
Dead fetuses/litter (mean %)	0	0	0	0
Resorption sites/litter (mean)	0.5	0.4	0.6	2.2**
Mean No. of live fetuses/litter	9	8.6	8.7	8.6
Mean fetal weights males (g)	3.6	3.6	3.7	3.6
Mean fetal weights females (g)	3.4	3.5	3.5	3.4
% of fetuses with external malformations	0	0.5	0	0
% of fetuses with visceral malformations	0	0	0	0
% of fetuses with skeletal malformations	0	1.9	0	2.6
% of fetuses with external variations	0	0	0	0
% of fetuses with visceral variations	8.8	8.7	13	23
% of fetuses with skeletal variations	96	96	97	100

** p<0.01 (Dunnett test or Kruskal-Wallis and Wilcoxon)