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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in compliance with GLP regulations

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995
Reference Type:
secondary source
Title:
SIDS for Propypelene Glycol Phenyl Ether - CAS No: 770-35-4 (major isomer - Secondary Alcohol), 4169-04-4 (minor isomer - Primary Alcohol), 41593-38-8 (commercial mixed isomer product). Oct. 2006
Author:
OECD
Year:
2006
Bibliographic source:
SIDS Initial Assessment Report For - SIAM 18- Paris, France, 20-23 April 2004

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1-phenoxypropan-2-ol
EC Number:
212-222-7
EC Name:
1-phenoxypropan-2-ol
Cas Number:
770-35-4
Molecular formula:
C9H12O2
IUPAC Name:
1-phenoxypropan-2-ol
Details on test material:
- Name of test material (as cited in study report): Protectol PP (CAS No 3860-05-4; test substance no 93/57)
- Physical state: colorless liquid
- Analytical purity: 99.9%
- Composition of test material, percentage of components: 1-phenoxypropan-2-ol) and 2-phenoxypropan-1-ol
- Stability under test conditions: verified by reanalysis
- Storage condition of test material: at room temperature
- Lot/batch No.: 18-2967

Test animals

Species:
rabbit
Strain:
Himalayan
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: 25-32 weeks; (Chbb :HM (outbred strain)) supplied by Dr. K. THOMAE GmbH, Biberach an der Riss, FRG,
- Age at study initiation: sexually mature, virgin Himalayan rabbits
- Weight at study initiation: 2.727 kg
- Fasting period before study:
- Housing: singly in type 12.2395.C stainless steel wire mesh cages supplied by DRAHT-BREMER GmbH, Marktheidenfeld, FRG (floor area about 3000 cm2 )
- Diet: Kliba maintenance diet type 23-341-4 for rabbits supplied by KLINGENTALMUEHLE AG, CH-4303 Kaiseraugst, Switzerland; ad libitum
- Water: tap water quality from water bottles; ad libitum
- Acclimation period: 5 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: emulsion in doubly distilled water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical investigations to determine the active ingredient content of the test substance were carried out before the beginning of the study (method: capillary gas chromatography)
Details on mating procedure:
- Impregnation procedure: artificial insemination
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
d 7-19 post insemination
Frequency of treatment:
daily, 7 d/wk
Doses / concentrations
Remarks:
Doses / Concentrations:
60, 180, 540 mg/kg per day
Basis:
actual ingested
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a range finding study (BASF AG study no. 20R0057/93018)

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS AND 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- 29 p.i.). The animals showing clinically evident signs of toxicity were observed several times a day, if necessary. A check was made twice daily on working days or once daily (Saturday, Sunday or on public holidays; days 0 - 29 p.i.).

BODY WEIGHT: Yes
- Time schedule for examinations: all animals were weighed on days 0, 2, 4, 7, 9, 11, 14, 16, 19, 21, 23, 25 and 29 p.i. The body weight change of the animals was calculated from these results.

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
On day 29 p.i., the surviving dams were sacrificed in randomized order by intravenous injection of a pentobarbital and the fetuses were removed from the uterus.
Dams showing signs of abortion were also sacrificed by intravenous injection of a pentobarbital. These animals as well as the contents of the uterus from these animals were investigated, if possible in the same way as at terminal sacrifice (exception: organ weights).
After the dams had been sacrificed, they were necropsied and assessed by gross pathology.
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 uterus, the ovaries, the liver and the kidneys were removed and the following data were recorded: weight of the liver (absolute and relative); weight of the kidneys (absolute and relative); weight of the unopened uterus; number of corpora lutea; number and distribution of implantation sites (classified as live fetuses and dead implantations): a) early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy); b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible); c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened).
Fetal examinations:
- External examinations: Yes; at necropsy each fetus was weighed and examined macroscopically for any external findings. Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes and fluids were examined. Individual placental weights were recorded.
- Soft tissue examinations: Yes; after the fetuses had been sacrificed by subcutaneous injection of a pentobarbital, the abdomen and thorax were opened in order to be able to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to assess the internal structure. The sex of the fetuses was determined by internal examination of the gonads.
- Skeletal examinations: Yes; After skinning, all fetuses were fixed in ethyl alcohol. After fixation for approx. 1- 5 days, the fetuses were removed from the fixative for a short while and a cross section of the heads from all intact fetuses was made in the parietal bone area using a scalpel. The two halves of the heads were carefully bent to allow a thorough examination of the brain. Subsequently, the fetuses were placed back into the fixative for further fixation. If heads of fetuses revealed severe findings (e.g. anophthalmia, microphthalmia, hydrocephalus, or cleft palate), the heads of these fetuses were severed from the trunk, fixed in BOUIN's solution and later processed and assessed according to WILSON's method. About 10 transverse sections were prepared per head. After the examination the heads treated in this way were discarded.
- Head examinations: Yes; after fixation in ethyl alcohol the skeletons (with the possible exception of the skulls) were stained according to a modified method of DAWSON, The stained skeletons were placed on an illuminated plate and examined, evaluated and assessed. After the examination the stained skeletons were retained by litter.
Statistics:
Examinations of dams and fetuses:
- The DUNNETT-Test was used for a simultaneous comparison of several dose groups with the control. The hypothesis of equal means was tested. This test was performed two-sided and was used for the statistical evaluation of the following parameters: food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), liver and kidney weights (absolute and relative), weight of the unopened uterus, number of corpora lutea, number of implantations, number of resorptions and number of live fetuses; proportion of preimplantation loss, postimplantation loss, resorptions and live fetuses in each litter; litter mean fetal body weight and litter mean placental weigh.
- FISHER’s Exact Test was used for a pairwise comparison of each dose group with the control for the hypothesis of equal proportions. This test was performed one-sided and was used for female mortality, females pregnant at terminal sacrifice and the number of litters with fetal findings.
- The WILCOXON Test was used for a comparison of each dose group with the control for the hypothesis of equal medians. This test was performed one-sided and was used for the proportion of fetuses with malformations, variations, retardations and/or unclassified observations in each litter.
Indices:
conception rate; preimplantation loss; postimplantation loss
Historical control data:
historical control data were available to allow comparison with concurrent controls

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
CLINICAL DATA
(1) Food intake, body weights:
Food intake of the high dose animals (at 540 mg/kg per day) was reduced up to 17% at beginning of the treatment (day 7-11), but reached or even exceeded that of controls thereafter (day 12-29). Food intake of animals receiving 60 or 180 mg/mg per day was not influenced. With regard to mean BW, no statistically significant differences were seen between controls and treated animals. In contrast, BWC of the high dose animals was significantly (day 7-9) or markedly reduced during day 9-14 p.i. (post insemination). During the entire treatment period (day 7-19 pi) these animals gained ca. 79% less than control animals. No such effect was seen in the groups receiving 60 or 180 mg/kg per day. No differences between test groups were seen with respect to corrected body weight gain, and no clear relation to dosing was observed.
(2) Mortalities, signs of toxicity:
No mortalities occurred. One high dose dam which aborted was sacrificed on day 28. From day 9-19 an increasing number of high dose animals showed apathy shortly after daily dosing. 14/15 animals were affected on day 16-19. Lateral position was seen in 6/15 animals on some days. These signs appeared shortly after dosing and persisted for several hours (until termination of working hours). The symptoms subsided overnight and animals were free of symptoms the next morning. None of the symptoms were seen after cessation of the treatment (day 20-29). One high dose doe showed no defecation from day 19-28 pi. It aborted on day 26 and was sacrificed on day 28 pi. Abortion rate is very low in this strain, thus abortion might be treatment-related. There were no clinical signs in the other does of the study.

TERMINAL NECROPSY FINDINGS
Organ weights, reproductive data:
- The mean gravid uterus weights did not differ between test groups. Test animals liver and kidney weights (relative and absolute) were not influenced by treatment. At necropsy, only spontaneous findings were noted; i.e. ulceration of the stomach (one control), small necrotic area in liver (one high dose), blind ending uterine horn (in 2 low dose animals).
- Conception rate was 93% in the control and 100% in the test groups.
- No substance-related biological relevant differences between groups were seen with respect to conception rate, mean number of corpora lutea and implantation sites or in the calculated values for pre- and postimplantation losses, the number of resorptions and viable fetuses. No dead fetuses were seen.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
540 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
No differences of biological relevance were seen between test groups with respect to fetal sex distribution, weight of fetuses or placentae weight. Any differences seen were within the range of biological variation of the historical data.

- External examination:
External malformations were only seen in one high dose fetus. Beside numerous external malformations (absence of the head; midline fissure of chest and abdominal wall; reduced number of digits on both forelimbs; shortened toes on both forelimbs and right hind limb) this fetus also showed soft tissue and skeletal malformations. The majority of the above mentioned malformations was already seen sporadically in this rabbit strain. External variations were seen in the high dose fetus mentioned above and in another medium dose fetus. These were (1) a flexure of the forelimb in the carpal joint and (2) rotation of one or two hindlimbs.

- Soft tissue examination:
In one high dose fetus, malformations of heart, thymus, lung, the great vessels and the gallbladder were seen. A septal defect of the heart was also seen in a medium dose fetus; a low dose fetus showed malformation of the gallbladder. Soft tissue variations were detected in each group including the control and occurred most frequently in the medium dose group. Variations included separated origin of the carotids, heart with traces of interventricular foramen, and hypoplasia of the gallbladder. As unclassified observation, one focal liver necrosis was seen in a low dose fetus.
- Skeletal malformations: No malformation was seen in the control fetuses (0/90); in the treated groups, 2/91 low dose fetuses (=2.2%), 2/100 medium dose (=2.0%) and 3/82 high dose fetuses (=3.7%) showed skeletal malformations. In the already mentioned fetus No. 5 these were correlates of the external findings (e.g. cleft sternum; digits absent). The other malformations were related to vertebral column and occurred in single fetuses without a clear relation to dosing. Almost all skeletal variations were observed in all groups without a clear relation to dosing, without biological relevant differences between the groups, and/or can be found at a comparable frequency in the historical control data. A statistically (but not significantly) increased incidence of accessory 13th rib in high dose fetuses was regarded as being treatment-related since the mean percentage of affected fetuses/litter (10.2%) is outside the historical control range (2.0%). Skeletal retardations were observed in all test groups in comparable frequencies.

The fetal findings were summarized and assessed as follows:

* MALFORMATIONS
1) The morphological examinations did not reveal dose-related, statistical significant increases in fetal external, soft tissue or skeletal malformations.
2) However, various malformations in the substance-treated groups occurred: a fetus (No. 5) showed a large variety of external, soft tissue and skeletal malformations. Taking into account the isolated nature of these findings and the fact that most of these malformations are contained in historical data for control fetuses of this rabbit strain, a relation to treatment can be excluded.
3) If this fetus No. 5 is excluded, only one low and one medium dose fetus showed soft tissue malformations (septal heart defect and agenesia of the gallbladder). Both malformations are assessed to be spontaneous, since they are also present in historical control data even at a higher incidence.
4) If the fetus No. 5 is excluded, skeletal malformations occurred in 6 treated fetuses, 2 of each dose group. These were vertebral column defects which are also present at even higher rates in the historical control data. Therefore, these findings are considered to be coincidental. 5) The overall malformation rate was not increased in a dose-related manner.
6) It was, however, significantly increased in the medium dose group receiving 180 mg/kg per day. Comparison with historical control data from previous studies with the same strain reveals that the mean percentage of affected fetuses/litter is in the same range as historical control animals (180 mg/kg per day: 3.4%; historical controls: 2.1-5.3%). Statistical significance in the present study resulted from an unexpectedly low incidence of malformations in control animals (0%) in the present study.

* VARIATIONS
Statistically significant increases were seen for:
1) One soft tissue variation (heart with interventricular foramen) at the intermediate and high dose levels,
2) For the overall rate of skeletal variations at the high dose, and
3) The overall rate of external, soft tissue and skeletal variations at medium and high dose.
- The mentioned soft tissue variation is not regarded as being substance-related. Statistical significance is due to an unexpectedly low incidence in the control group, as evidenced by comparison with historical control data.
- The increase of skeletal variations in high dose fetuses is mainly caused by an increased occurrence of a 13th rib. Numerous publications dealing with this phenomenon (e.g. Kehra; Kimmel & Wilson; Wickramaratne), consider this as manifestation of an unspecific stress, rather than as a teratogenic effect. In this study, maternal toxicity was observed in the high dose group (reduced food intake, reduced body weight gain, adverse clinical symptoms). Therefore, the increased occurrence of accessory 13th rib at 540 mg/kg per d is assessed as an embryotoxic effect representing a manifestation of a non-specific stress on the does. It is not interpreted as a teratogenic effect of the test substance. Therefore, the significantly increased rates of total variations in medium and high dose fetuses is misleading. The facts discussed above (i.e. unexpectedly low incidence in controls and interpretation of 13th rib) need instead to be taken into account.

* SKELETAL RETARDATIONS
No substance-related, biologically relevant difference between the groups occurred. The statistically significant increase in total retardations at 60 mg/kg per day is considered random since it is not dose-related and this degree of variability has been observed in previous studies using this strain of rabbits.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
> 540 mg/kg bw/day
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

CONCLUSION

Under the conditions of this study, the administration of test substance to pregnant Himalayan rabbits during organogenesis induced overt maternal toxicity at 540 mg/kg body weight/day, but was not toxic to the does at 60 and 180 mg/kg body weight/day. Maternal toxicity at the highest dose level was substantiated by reduced food consumption, impairments in body weight gain and adverse clinical symptoms (e.g. apathy/lateral position).

Signs of developmental toxicity occurred only at the highest dose level (540 mg/kg body weight/day) in the form of an increased occurrence of skeletal variations (predominantly accessory 13th rib(s)); however, no substance-induced teratogenic effects were observed up to and including the dose of 540 mg/kg body weight/day.

At 60 and 180 mg/kg body weight/day no influence on the gestational parameters and no signs of developmental toxicity, especially no substance-induced indications of teratogenicity, were observed.

For this prenatal toxicity study in Himalayan rabbits, the no observed adverse effect level (NOAEL) concerning maternal and developmental toxicity is 180 mg/kg body weight/day.

Applicant's summary and conclusion

Conclusions:
For this prenatal toxicity study in Himalayan rabbits, the no observed adverse effect level (NOAEL) concerning maternal and developmental toxicity is 180 mg/kg body weight/day.
Executive summary:

Under the conditions of this study, the administration of test substance to pregnant Himalayan rabbits during organogenesis induced overt maternal toxicity at 540 mg/kg body weight/day, but was not toxic to the does at 60 and 180 mg/kg body weight/day. Maternal toxicity at the highest dose level was substantiated by reduced food consumption, impairments in body weight gain and adverse clinical symptoms (e.g. apathy/lateral position). Signs of developmental toxicity occurred only at the highest dose level (540 mg/kg body weight/day) in the form of an increased occurrence of skeletal variations (predominantly accessory 13th rib(s)); however, no substance-induced teratogenic effects were observed up to and including the dose of 540 mg/kg body weight/day. At 60 and 180 mg/kg body weight/day no influence on the gestational parameters and no signs of developmental toxicity, especially no substance-induced indications of teratogenicity, were observed. For this prenatal toxicity study in Himalayan rabbits, the no observed adverse effect level (NOAEL) concerning maternal and developmental toxicity is 180 mg/kg body weight/day.