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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
Study period:
13. May 2005 - 23. Jun. 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identity : BENZOIC ACID ISONONYLESTER
Labelled as : ISONONYLBENZOAT
Batch no. : 04/3945-3947
Expiry date : 30 June 2006
Appearance : Clear liquid
Storage conditions : Room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy.
- Age at study initiation: 9 weeks old
- Weight at study initiation: 202-225 g
- Fasting period before study: no
- Housing: The animals were housed in a limited access rodent facility. clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent paper, which was inspected and changed at least 3 times per week. During the mating period, the rats were housed on the basis of 1 male to 1 female in clear polycarbonate cages measuring 43x27x18 cm instead of 43x27x15 cm as specified in the original study protocol with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material, which was inspected and changed daily.
- Diet: 4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy ad libitum
- Water: ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The test item was administered orally by gavage at a dose volume of 4 ml/kg body weight. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal. All animals were dosed once a day from Day 0 through Day 19 post coitum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to commencement of treatment the proposed formulation procedure was checked by chemical analysis to confirm that the method was acceptable The stability of the formulations was found to be 24 hours at room temperature. These analyses were performed in the present study (for high dose level) and in the RTC Study No.: 26960 (for low dose level). Samples of the formulations prepared during the first and last weeks of the study were analysed to check the concentration and homogeneity. Results of all the analyses were within the limits of acceptance. On week 1 of treatment, for group 4, (concentration of 250 mg/ml) a repetition of analysis was performed. The results were within the limit of acceptance.
Details on mating procedure:
The females were paired with male rats. Females were paired one to one in the home cage of the male and left overnight. The day of mating, as judged by the presence of sperm in the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post-coitum). Full mating records were maintained. Vaginal smears were taken daily in the morning from the day after pairing until a positive identification of mating was made.
Duration of treatment / exposure:
Dose levels were selected in consultation with the Sponsor based on a previous study in rats (RTC Study number 40330EXT). All animals were dosed once a day from Day 0 through Day 19 post coitum.
Frequency of treatment:
All animals were dosed once a day from Day 0 through Day 19 post coitum.
Duration of test:
28 days
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected in consultation with the Sponsor based on a previous study in rats (RTC Study number 40330EXT).
- Rationale for animal assignment (if not random): random
- Other:

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately midday.
- Cage side observations: Full records were maintained for all measurements and observations.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All clinical signs were recorded daily for individual animals. Each animal was observed daily from allocation until sacrifice.


BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: All animals were killed on Day 20 post coitum and necropsied
- Organs examined: The clinical history of the animal was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). All changes were noted and the abnormalities preserved in 10% buffered formol saline (except eyes, optic nerves and Harderian glands which were fixed in Davidson's fluid). The ovaries and uteri were examined to determine:
• number of corpora lutea;
• number of implantation sites;
• weight of intact gravid uterus;
• number, sex and weight of all live foetuses;
• number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing);
• number and distribution of intra-uterine deaths;
• gross evaluation of placenta.

OTHER:
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:
Fetal examinations:
All live foetuses were examined externally. Approximately one-half of the foetuses (i.e., routinely, every second live foetus) in each litter was preserved in Bouin's solution for subsequent fixed-visceral examination. The remaining foetuses were eviscerated after which the carcasses were fixed in 95% (v/v) ethanol for subsequent skeletal examination. Skeletal and soft tissue examinations were performed in all groups.
Structural deviations were classified as follows:
Malformations : major abnormalities that are rare and/or affect the survival or health of the species under investigation.
Anomalies : minor abnormalities that are detected relatively frequently.
Variants : A change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. This might include a delay in growth or morphogenesis that has otherwise followed a normal pattern of development.
Statistics:
For continuous variables the significance of the difference amongst group means was assessed by analysis of variance. Differences between treated groups and the control group were assessed by Dunnett’s test using a pooled error of variance. The homogeneity of the data was assessed by Bartlett’s Test before Dunnett’s Test was performed. If the data were found to be inhomogeneous, a modified t-test (Cochran and Cox) was applied.
The criterion for statistical significance was p<0.05 and 0.01.
The non-parametric Kruskal-Wallis analysis of variance was used for all other parameters. Intergroup differences between the control and the treated groups were assessed by the non-parametric version of the Williams' test.
The criterion for statistical significance was p<0.05.
The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
An increased incidence of scabs was noted in the high dose females when compared to the control group. Statistically significant decreases in body weight were observed in the high dose group on Days 18 and 20 post coitum. Changes in body weight gain were also noted in the high dose group. A statistically significant reduction in terminal body weight, uterus weight, absolute weight gain and litter and mean foetal weight was observed in the high dose group.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
A general retardation of the ossification of the skull, 5th and 6th sternebrae, thoracic centra, pubis bones and sacral arches was observed in the high dose group at skeletal examination. These changes were considered to be a consequence of the presence of the foetuses with a lower foetal weight and due to the signs of marginal maternal toxicity observed in the high dose group.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
On the basis of these results the mid- dose level (300 mg/kg/day) could be considered as the NOAEL for both maternal and foetal toxicity.
Executive summary:

The effects of BENZOIC ACID ISONONYLESTER on pregnancy and embryo-foetal development were assessed in the rat during gestation. Three groups of 24 mated female rats were used in the study and received BENZOIC ACID ISONONYLESTER at dosages of 100, 300 and 1000 mg/kg/day, respectively. A similarly constituted group (Group 1) received the vehicle alone (corn oil) and acted as a control. The animals received the test item or the vehicle from Day 0 to Day 19 post coitum. An increased incidence of scabs was noted in the high dose females when compared to the control group. Statistically significant decreases in body weight were observed in the high dose group on Days 18 and 20 post coitum. Changes in body weight gain were also noted in the high dose group. A statistically significant reduction in terminal body weight, uterus weight, absolute weight gain and litter and mean foetal weight was observed in the high dose group. A general retardation of the ossification of the skull, 5th and 6th sternebrae, thoracic centra, pubis bones and sacral arches was observed in the high dose group at skeletal examination. These changes were considered to be a consequence of the presence of the foetuses with a lower foetal weight and due to the signs of marginal maternal toxicity observed in the high dose group. On the basis of these results the mid- dose level (300 mg/kg/day) could be considered as the NOAEL for both maternal and foetal toxicity.