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Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
other: read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Remarks:
Justification for Read Across is reported in the endpoint summary and in the Category Justification Report attached to the Section 13 of this dossier.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2021

Materials and methods

Principles of method if other than guideline:
The dose-range finding experiment was performed with 3 groups of treated pregnant female rabbits and a control group to find appropriate dose for the OECD 414 study .
The purpose of this preliminary study was to investigate the effects of the substance on pregnancy and embryo-foetal development in New Zealand rabbit, following oral administration from Days 6 through Day 28 post coitum at dose levels of 100 and 300, and 750 mg/kg and the dose volume of 10 ml/kg. The control group animals received softened water at the same dose volume. During the dosing period, food was removed each day from 3 hours
before and up to 3 hours after administration.
The emptying of the stomach after 6 hours of fasting was also evaluated through the weight of the full and empty stomach.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
OB 4-MSA
IUPAC Name:
OB 4-MSA
Test material form:
solid: pressed powder

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
A minimum of 34 sexually mature virgin female New Zealand White rabbits SPF, 13 weeks old
and at least 2.5 kg body weight, were ordered from Charles River Laboratories Italia S.r.l. The
males used were of the same strain from the same supplier and were at least 25 weeks of age
and more than 3.5 kg in body weight.
Sex: females (males – only for mating)
Total number of animals: 6 females and 6 males per group
Acclimatization: 20 days
Animal room controls were set to maintain temperature and relative humidity at 19°C ± 2°C and 55% ± 15%, respectively.
There were approximately 15-20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.

Drinking water was supplied ad libitum to each cage via water bottles.
A commercially available laboratory rabbit diet (2RB15, Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy) was offered ad libitum before the start of dosing.
During the dosing period, food was removed each day from 3 hours before and up to 3 hours after administration.

On the day of allocation (Day 0 post coitum), all females were weighed. The rabbits were allocated to the groups by stratified randomisation to give approximately equal initial group mean body weights. The females were identified within the study by an ear tattoo and reassigned to cages in the study room. The cages were identified by a label recording the study number, animal numbers and details of treatment. The cages were arranged in racks to minimise possible environmental differences between groups.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
During the dosing period, food was removed each day from 3 hours before and up to 3 hours after administration.

- Dose volume: 10 mL/kg body weight
-Control animals received the vehicle alone at the same dose volume

The required amount of test item was dissolved in the vehicle. The preparations were made daily (concentrations of 10, 30 and 75 mg/mL) and protected from light. Concentrations were calculated and expressed in terms of test item as supplied.
Analytical verification of doses or concentrations:
no
Details on mating procedure:
Females were introduced to fertile males obtained from the same supplier. Each female remained with the male for at least 1 hour after successful mating was observed. The day successful mating was detected and was considered Day 0 post coitum (or gestation Day 0).
Duration of treatment / exposure:
Females at final sacrifice were treated once a day from Day 6 through Day 28 post coitum.
Females at interim sacrifice were treated once a day from Day 6 through Day 28 post coitum (control group) and from Day 6 through Day 25/26 post coitum (Highest dose).
Frequency of treatment:
daily - 7 days per week
Duration of test:
Females at final sacrifice were treated once a day from Day 6 through Day 28 post coitum.
Females at interim sacrifice were treated once a day from Day 6 through Day 28 post coitum (Group 1) and from Day 6 through Day 25/26 post coitum (Group 4).
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6
Control animals:
yes
yes, concurrent vehicle
Details on study design:
Three females randomly selected from Group 1 (control) and three females from Group 4 (high dose) were killed between Days 25 and 28 post coitum for evaluation of the empyting of the stomach after 6 hours of fasting.

Dose levels of 100, 300 and 750 mg/kg/day were selected based on information from a previous study on another members of category, where an excessive mortality and maternal toxicity was observed at 800 mg/kg/day.

Examinations

Maternal examinations:
- Mortality
Throughout the study, all animals were checked early in the morning 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 mid-day. This allowed post mortem examinations to be carried out during the working period of that day.
- Clinical signs
All clinical signs were recorded for individual animals. Once before commencement of treatment and once daily during treatment, each animal was observed and any clinical signs was recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.
- Body weight
Each animal was weighed on the day of allocation to treatment group (Day 0 post coitum) and on Days 6, 9, 12, 15, 18, 21, 24, 27 and 29 post coitum or the day of sacrifice.
- Abortions
Animals that aborted were killed by an intravenous injection of a suitable euthanasia agent (Pentobarbital overdose or Tanax®. Tanax was injected in sedated animals) on the same day that the abortion was detected. The females were subjected to a detailed macroscopic examination and the number of corpora lutea and implantation sites were recorded.
- Necropsy
All animals, including those killed for humane reasons, were subjected to necropsy and the number of implantations and corpora lutea was recorded if possible. 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 retained in an appropriate fixative.
- gravid uterus weight, absolute weight gain (terminal body weight minus gravid uterus weight minus body weight at Day 6 post coitum), litter size, intra-uterine deaths, corpora lutea count and pre- and postimplantation loss were calculated.
Ovaries and uterine content:
– Gravid uterine weight (not obtained from animals found dead, killed during the study or with total resosrption of both uterine horns);
– number of corpora lutea for pregnant animals;
– number of implantations for pregnant animals;
– number, sex and weight of all live foetuses;
– number and sex of dead foetuses (foetuses at termwithout spontaneous movements and breathing);
– number of intra-uterine deaths;
– gross evaluation of placentae.
Fetal examinations:
Pathological examination of foetuses
The number of foetuses affected with structural deviations and the corresponding litter percentage were calculated.
All derived values (e.g. means, percentages, ratios) first were calculated within the litter and the group values derived as a mean and standard deviation of individual litter values.
Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters.
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of theWilliams test.
Indices:
Pre-implantation loss was calculated as a percentage from the formula:
Pre impl. Loss%= no. of corpora lutea−no. of implantations / no. of corpora lutea ×100
Post-implantation loss was calculated as a percentage from the formula:
Post impl. Loss%= no. of implantations−no. of live young / no. of implantations ×100
Total implantation loss was calculated as a percentage from the formula:
Total impl. Loss%= no. of corpora lutea−no. of live foetuses / no. of corpora lutea ×100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Blood loss (recorded as red staining) and/or foetuses in the cage tray were recorded in the three high dose females and one mid-dose female, sacrificed for humane reasons. Inappetence, reduced water consumption and reduced faeces were also observed in 2 out of these 3
high dose females and pallor in the mid-dose female.
No significant signs were observed in the control and low dose groups with the exception of reduced faeces in one control female and brown staining of the perianal region in one low dose female. No other signs were observed throughout the study.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two high dose females were sacrificed for humane reasons following evidence of blood in the cage. At necropsy they had total early resorption. An additional high dose female and one mid-dose female were sacrificed for abortion.
The most relevant changes observed at post mortem examination in the three high dose females sacrificed were thickened caecum and/or stomach, brown fluid content of caecum, white content or red colour of trachea and in one instance also dark red gelatinous content of uterus. No relevant changes were observed in the female rabbit of the mid-dose group
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight and body weight gain were comparable between the controls and all treated groups.
Description (incidence and severity):
Inappetence, reduced water consumption and reduced faeces were also observed in 2 out of these 3 high dose females
Description (incidence and severity):
Inappetence, reduced water consumption and reduced faeces were also observed in 2 out of these 3 high dose females
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The uterus weight, the corrected body weight (terminal body weight minus the uterus weight) and the absolute weight gain, calculated subtracting the uterus weight and the body weight at gestation Day 6 from the terminal body weight, did not statistically differ between groups
of females at term (Groups control to mid-dose).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Unscheduled deaths
Three high dose females and one mid-dose female were sacrificed for humane reasons from Day 17 to Day 25 post coitum, following evidence of blood in the cage and, in the case of one high dose female and one mid-dose female for abortion. The most relevant changes observed at post mortem examination in the three high dose females were thickened caecum and/or stomach, brown fluid content of caecum, white content or red colour of trachea and in one
instance also dark red gelatinous content of uterus. No relevant changes were observed in the female rabbit of the mid-dose group.
Interim and final sacrifice
No relevant changes were observed in the female rabbits sacrificed at interim or that completed the scheduled test period when compared to the controls
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Three control and 3 high dose females were sacrificed at interim before Day 29 post coitum for evaluation of the emptying of the stomach after 6 hours of fasting.
No gastric emptying was found in the animals examined interim after 6 hours of fasting. In addition, a statistically significant increase of relative full stomach weight (approximately 53%) was noted in the high dose female when compared to the controls

Maternal developmental toxicity

Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
An high dose female and one mid-dose female were sacrificed for abortion
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Implantations and pre-implantation loss did not statistically differ between groups (Groups 1 to 4) and the valueswere within the range of historical control data.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
Total resorption at early stage was observed only in high dose females.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
Two high dose females were sacrificed for humane reasons following evidence of blood in the cage. At necropsy they had total early resorption.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
At external examination of foetuses performed on Groups control to mid-dose, a total of 23 small foetuses were recorded: 12 out of 28 foetuses in the control group, 1 out of 45 in the low dose group and 10 out of 41 in the mid-dose group.
Although the incidence did not follow a dose relation pattern, the percentage of litters with small foetuses involved in the control and mid-dose group (67% and 60%, respectively) was outside the historical control data.
Details on maternal toxic effects:
No treatment related effects were noted in litter data of low and mid-dose group compared to the control. The high percentage of litters with small foetuses noted in the control and mid-dose group could be considered in favour of an effect due to food deprivation.

Effect levels (maternal animals)

Dose descriptor:
LOAEL
Effect level:
750 mg/kg bw/day (nominal)
Basis for effect level:
clinical signs
gross pathology
mortality
Remarks on result:
other:
Remarks:
Abortion was considered due the maternal toxicity and not a direct effect of the test item on the pregnancy outcome.

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At external examination of foetuses performed on Groups control to mid-dose, a total of 23 small foetuses were recorded: 12 out of 28 foetuses in the control group, 1 out of 45 in the low dose group and 10 out of 41 in the mid-dose group.
Although the incidence did not follow a dose relation pattern, the percentage of litters with small foetuses involved in the control and mid-dose group (67% and 60%, respectively) was outside the historical control data.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined

Any other information on results incl. tables

The same table is also found in the information panel.

  dose (mg/kg) notes
total females = 6 per dose 0 100 300 750
Unscheduled Death(s) 0 0 1 3 During the in life phase of the study, 3 high dose animals were sacrificed on gestation Day 17 or 19 and one mid-dose female was sacrificed on gestation Day 25. All animals were sacrificed due to the presence of red staining or foetuses on the cage tray.
Health conditions:          
Blood loss (recorded as red staining) 0 0 1 3 the animals were sacrificed for humane reasons from Day 17 to Day 25 post coitum, following evidence of blood in the cage
Inappetence and reduced water consumption 0 0 0 2  
Reduced faeces 1 0 0 2  
Body weight (kg) - Group mean data 3.873 3.857 3.853 3.820 At day 24 post coitum. The body weight and body weight gain were comparable between the controls and all treated groups.
Haematological examination not performed not performed not performed not performed  
Necropsy evaluation at interim and final sacrifice no effects no effects no effects effects As fornscheduled deaths:
Three high dose females and one mid-dose female were sacrificed for humane reasons from Day 17 to Day 25 post coitum, following evidence of blood in the cage and, in the case of one high dose female and one mid-dose female for abortion. The most relevant changes observed at post mortem examination in the three high dose females were thickened caecum and/or stomach, brown fluid content of caecum, white content or red colour of trachea and in one
instance also dark red gelatinous content of uterus. No relevant changes were observed in the female rabbit of the mid-dose group.
Number of pregnant/non pregnant  6/0 6/0 6/0 6/0  
Number of Dams/Fetus 6/28 6/45 6/41 n.a.  
Gravid uterus Weight (mean- g) 451.88 446.69 407.36 n.a.  
Dead foetuses 0 0 0 n.a.  
Mean foetal weight (g) 42.07 38.62 28.25 n.a.  
Corpora lutea (mean) 9.67  8.50  9.67 10.00   
Implantations (mean) 9.33 7.67 9.00 9.83  
Total resoptions (mean) 0.00 0.17 0.20 3.33 Total resorption at early stage was observed only in high dose females.
Description and incidences of malformations and main variations small foetuses  n.a. small foetuses  n.a. At external examination of foetuses performed on Groups 1 to 3, a total of 23 small foetuses were recorded: 12 out of 28 foetuses in the control group, 1 out of 45 in the low dose group and 10 out of 41 in the mid-dose group.
Although the incidence did not follow a dose relation pattern, the percentage of litters with small foetuses involved in the control and mid-dose group (67% and 60%, respectively) was outside the historical control data.

Applicant's summary and conclusion

Conclusions:
On the basis of the results, it can be concluded that the test item at the dose level of 750 mg/kg/day is not tolerated by pregnant rabbits, as demonstrated by the clinical signs and the changes noted at the gastrointestinal tract at necropsy. As a consequence of the gastrointestinal disturbance the animals aborted or were sacrificed for humane reasons.

No gastric emptying was found in the animals examined after 6 hours of fasting during the treatment period. In addition, a possible slowdown in gastric emptying in females dosed at 750 mg/kg/day was highlighted by the significant increase in these females of the weight of the full stomach.
Executive summary:

Based on the results of this preliminary study, maternal toxicity at the dosage of 750mg/kg/day was confirmed by the findings noted at post mortem examination in the high dose females sacrificed. Abortion was considered due the maternal toxicity and not a direct effect of the test item on the pregnancy outcome.

Therefore, the high dose level should not exceed 300 mg/kg/day in the subsequent reproductive toxicity studies in rabbits.

No gastric emptying was found in the animals examined after 6 hours of fasting during the treatment period. In addition, a possible slowdown in gastric emptying in females dosed at 750 mg/kg/day was highlighted by the significant increase in these females of the weight of the full stomach.