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Toxicological information

Developmental toxicity / teratogenicity

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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: No OECD guideline or GLP defined.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1997

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
other: Developmental toxicity study in rats.
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Phthalic acid
EC Number:
201-873-2
EC Name:
Phthalic acid
Cas Number:
88-99-3
Molecular formula:
C8H6O4
IUPAC Name:
benzene-1,2-dicarboxylic acid
Details on test material:
Test substance Phthalic Acid (PA) (99.5% pure, Aldrich, Milwaukee, WI).

Test animals

Species:
rat
Strain:
Wistar Kyoto (WKY)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: about 12 weeks
- Weight at study initiation:
- Fasting period before study:
- Diet (e.g. ad libitum): basal diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 +- 1°C
- Humidity (%): 55 +-5%
- Air changes (per hr): air-conditioned room
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: diet
Details on exposure:
The pregnant rats were fed a diet containing phthalic acid (PA, 99.5% pure, Aldrich, Miwaukee, WI) at a dose of 1.25, 2.5, or 5.0% ad libitum on day 7 through to day 16 of pregnancy.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
The diet containing PA was prepared fresh weekly. A predetermined amount of PA was weighed and added to a small aliquot of ground basal diet and handblended. This premix was then added to a preweighed ground basal diet and blended with mill (Irie Shokai, Tokyo, Japan) for 30 min. The control rats were fed a basal diet only ad libitum.
Details on mating procedure:
Virgin female rats, about 12 weeks old, were mated overnight with male rats. The day when sperm were detected in the vaginal smear was considered to be day 0 of pregnancy. The pregnant rats were distributed on a random basis into four groups of 11 pregnant rats each housed individually.
Duration of treatment / exposure:
The pregnant rats were fed a diet ad libitum on day 7 through to day 16 of pregnancy.
Frequency of treatment:
continuous via food
Duration of test:
The pregnant rats were sacrified on day 20 of pregnancy.
Doses / concentrationsopen allclose all
Dose / conc.:
1.25 other: % in diet
Remarks:
daily test material intake determined with 1021 +- 52 mg/kg bw/day
Dose / conc.:
2.5 other: % in diet
Remarks:
daily test material intake determined with 1763 +- 163 mg/kg bw/day
Dose / conc.:
5 other: %
Remarks:
daily test material intake determined with 2981 +- 270 mg/kg bw/day
No. of animals per sex per dose:
11
Control animals:
yes
Details on study design:
The pregnant rats were fed a diet containing PA (99.5% pure, Aldrich, Milwaukee, WI) at a dose of 1.25, 2.5, or 5.0% ad libitum on day 7 through to day 16 of pregnancy. The administration in the feed was selected because of necessity to expose to large amount of PA and slight solubility of PA in water and oil (Budavari et al.,1996). This method for administration is useful with agents that are to be given in large amounts or are difficult to dissolve in vehicles that would be tolerated in other treatment routes (Wilson, 1965).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Average daily intake of PA was calculated by the method described by Tyl et al. (1988).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
Ovaries and uterine content:
The peritoneal cavity and uterus were opened and the numbers of live and dead fetuses and resorptions were counted.
The gravid uterus was removed and the rats weighed again. The adjusted weight gain, i.e. maternal weight gain throughout pregnancy corrected for gravid uterine weight, was calculated.
Fetal examinations:
The live fetuses removed from the uterus were sexed, weighed and inspected for external malformations and malformations within the oral cavity. Approximately two-thirds of live fetuses in each litter, randomly selected, were fixed in 99% ethanol, stained alizarin red S (Kawamura et al., 1990) and examined for skeletal malformations. The remaining live fetuses in each litter were fixed in Bouin’s solution and examined for internal malformations using the free-hand razor blade sectioning method of Wilson (1965).
Statistics:
Statistical analysis of the offspring data was carried out using the litter as a unit. Analysis of variance and Dunnett’s multiple comparison test, Kruskal–Wallis test and Mann–Whitney test or Fisher’s exact test were used as appropriate. The 0.05 level of probability was used as the criterion for significance.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
see Table 1
Mortality:
no mortality observed
Description (incidence):
see Table 1
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see Table 1: The maternal body weight gain on days 7–16 in the 2.5 and 5.0% groups and the adjusted weight gain, which indicates the net weight gain of maternal rats, in the 5.0% group were significantly lower than those in the control group. The maternal body weight gain on days 16–20 in the 5.0% group was significantly higher than that in the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see Table 1: A significantly decreased food consumption on days 7–16 in the 2.5 and 5.0% groups and a significantly increased food consumption on days 16–20 in the 1.25, 2.5 and 5.0% groups were found. The average daily intakes of PA were 1021 mg/kg for the 1.25% group, 1763 mg/kg for the 2.5% group, and 2981 mg/kg for the 5.0% group.

Maternal developmental toxicity

Description (incidence and severity):
The reproductive findings in rats given dietary phthalic acid (PA) on day 7 through day 16 of pregnancy are shown in Table 2. No significant differences between the PA-treated groups and the control group were detected in the numbers of corpora lutea per litter, implantations per litter, resorptions and dead fetuses per litter and live fetuses per litter, incidence of postimplantation loss per litter, and sex ration of live fetuses.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
ca. 1 021 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
ca. 1 763 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
see Table 2: The weight of male fetuses in the 5.0% group was significantly lighter than that in the control group, although no significantly decreased weight of female fetuses was found in any PA-treated groups.
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Description (incidence and severity):
see Table 3: A few types of skeletal variations in the vertebrae and sternebrae were observed in the control and PA-treated groups.
Visceral malformations:
no effects observed

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 763 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: body weight male fetuses

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Maternal findings in rats given dietary PA on days 7 -16 of pregnancy:

            PA (%)
   0 (control)  1.25  2.5  5.0
 No. of pregnant rats  11  11  11  11
 No. of dead pregnant rats  0  0  0  0
 Initial body weight (g)  242+10 243+16   243+12  244+16
 Body weight gain during pregnancya)        
 Days 0 -7  24+6  26+10  31+6  27+7
 Days 7 -16  49+8  54+8  40+5*  20+12**
 Days 16 -20  41+9  40+8  47+11  57+16**
 adjusted weight gainb)  50+11  47+10  42+10  30+12**
 Food consumption during pregnancy (g)a)        
 Days 0 -7  138+8  140+15  145+13  138+8
 Days 7 -16  198+11  197+10  173+17**  145+13**
 Days 16 -20  88+10  98+4**  101+6**  120+7**
 Daily intake of PA (mg/kg)a,c)  0  1021+52  1763+163  2981+270

a)Values are given as mean+S.D:

b)Adjusted weight gain refers to maternal weight gain excluding the gravid uterus.

c)[Food consumotion on days 7 -16/9)x%PA]/body weight on day 7.

*,**Significantly different from the control, P<0.05 and P<0.0.1, respectively.

Table 2: Reproductive findings in rats given dietary PA on days 7 -16 of pregnancy:

            PA (%)
   0 (control)  1.25 2.5  5.0
 No. of litters  11  11  11  11
 No. of corpora lutea per littera)  14.3 +1.3  15.3 +1.6  15.7 +2.3  15.7 +1.5
 No. of implantations per littera)  13.1 +2.1  14.0 +1.7  14.3 +3.3  13.8 +3.4
 No. of litters totally resorbed  0  0  0  0
 No. of resorbtions and dead fetuses per littera)  1.6 +1.6 1.3 +1.1   0.9 +1.0  1.3 +1.4
 % Postimplantation loss per litterb)  14.2  9.3  5.8  8.7
 No. of live fetuses per littera)  11.5 +3.0  12.7 +2.2  13.4 +3.0  12.5 +3.4
 Sex ratio of live fetuses (male/female)  60/66  61/79  83/64  62/76
 Body weight of live fetuses (g)a)        
 Male  4.19 +0.13  4.15 +0.07  4.20 +0.18  4.03 +0.18*
 Female  3.92 +0.16  3.95 +0.13  3.92 +0.15  3.82 +0.14

a) values are given as mean + S.D.

b) (No. of resorbtions and dead fetuses/no. of implantations)x100.

* Significantly different from the control, P<0.05.

Table 3: Morphological findings in fetuses of rats given dietary PA on days 7 -16 of pregnancy:

            PA (%)
   0 (control)  1.25  2.5  5.0
 external examination      
  No. of fetuses (litters) examined  126 (11) 140 (11)  147 (11)  138 (11)
  No. of fetuses (litters) with malformations  0  0  0  0
 Skeletal examination        
    No. of fetuses (litters) examined  84 (11)  94 (11)  97 (11)  92 (11)
    No. of fetuses (litters) with malformations  0  0  0
    No. of fetuses (litters) with variations  4 (3)  5 (4)  4 (3)  10 (6)
    No. of fetuses (litters) with:        
  Splitting of thoracic vertebral bodies  1 (1)  0  0  0
  Asymetric of sternebrae  3 (3)  4 (3)  4 (3)  7 (6)
  Splitting of sternebrae  0  1 (1)  0 5 (3) 
  Degree of ossification        
  No. of ossification centers of caudal vertebraea)  5.5 +0.3  5.3 +0.3  5.4 +0.3  5.1 +0.2*
  No. of sternebraea)  5.9 +0.1  6.0 +0  6.0 +0.1  5.9 +0.1
 Internal examination        
  No. of fetuses (litters) examined  42 (11)  46 (11)  50 (11)  46 (11)
    No. of fetuses (litters) with malformations  0  0  0  0

a) Values are given as mean +S.D.

* Significantly different from the control, P<0.01.

Applicant's summary and conclusion

Executive summary:

The developmental toxicity of phthalic acid was investigated in a developmental toxicity study. Groups of eleven pregnant Wistar rats were fed a diet containing phthalic acid at a dose of 0, 1.25, 2.5, or 5.0 % ad libitum on GD 7 - GD 16 ( average daily intake: 0, 1021, 1763, 2981 mg/kg bw/day). The pregnant rats were observed daily for evidence of clinical signs of toxicity, maternal body weight and food consumption, and were sacrificed on day 20 of pregnancy. The peritoneal cavity and uterus were opened and the numbers of live and dead fetuses and resorptions were counted. The gravid uterus was removed and the rats weighed again. The adjusted weight gain, i.e. maternal weight gain throughout pregnancy corrected for gravid uterine weight, was calculated. The live fetuses removed from the uterus were sexed, weighed and inspected for external malformations and malformations within the oral cavity. Approximately two-thirds of live fetuses in each litter, randomly selected, were stained with alizarin red S and examined for skeletal malformations. The remaining live fetuses in each litter were fixed in Bouin¿s solution and examined for internal malformations. Maternal toxicity occurred in the 2.5 and 5.0 % groups as demonstrated by decreases in the adjusted maternal bodyweight gain (maternal bw gain excluding the gravid uterus; 30, 42, or 50 g for the 5, 2.5, or control group, respectively) during the administration period. No significant changes in maternal parameters were found in the 1.25 % group (adjusted body weight gain 47 g). No deaths or clinical signs of toxicity were noted in any group. No significant changes induced by phthalic acid were detected in the incidence of postimplantation loss, number and sex ratio of live fetuses. No fetuses with external, skeletal and internal malformations were found in any group. Significant decreases in the weight of male fetuses and decreased numbers of ossified centers of the caudal vertebrae were found only in the 5.0 % group, where significant maternal toxicity also was observed. Morphological examinations of fetuses revealed no evidence of developmental toxicity (NOAEL, maternal toxicity: 1.25 % in feed = 1021 mg/kg bw/day; NOAEL, developmental toxicity: 2.5 % in feed = 1763 mg/kg bw/day).