REAKTIV-ORANGE DYPR 1410

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2001/2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Structural Analogue 01

IUPAC Name:

Structural Analogue 01

Test material form:

solid: particulate/powder

Details on test material:

See below

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

- Source: Harlan Winkelmann GmbH, Gartenstrasse 27, 33178 Borchen, Germany
- Age at study initiation: approximately 8 - 10 weeks
- Housing: single
- Diet (ad libitum): sniff R/M-Z (V1324)
- Water (ad libitum): tap
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 16-20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionised

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The test item was dissolved daily in deionized water in concentrations of 12.5 mg/mL, 50 mg/mL and 200 mg/mL.
Application volume : 5 mL/kg bw.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

First and last week of treatment

Details on mating procedure:

Virgin female animals in the pre-oestrus or oestrus phase were mated overnight with sexually mature males in the ratio 1 male : 1 female and were caged individually after the detection of sperm in vaginal smears. The day of sperm detection was defined as day 0 of gestation. Pregnancy was confirmed at necropsy by the detection of implantation sites or normally developed corpora lutea.

Duration of treatment / exposure:

day 6 to 19 of gestation

Frequency of treatment:

daily

Duration of test:

cesarean section on day 20 of gestation

No. of animals per sex per dose:

23

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a preliminary study groups of 6 mated female Sprague Dawley rats receivedthe test item orally by gavage once daily at the dose levels of 1000 mg/kg body weight/day from day 6 - 19 of pregnancy and were sacrificed on day 20 of pregnancy.
No compound-related clinical signs were observed. at the dose of 1000 mg/kg body weight. Body weight development and food consumption were considered not to be affected, based on the limited number of animals tested. No abnormalities were observed at necropsy of the animals. No abnormalities were detected at caesarean section. Embryofetal development was not impaired (Report No.: PT01-0026).
Based on the results of this study, the dose levels of 0 mg/kg bdwt, 62.5, 250, and 1000 mg/kg bw/day were selected for the present study.

Examinations

Maternal examinations:

Body weight
Animals were weighed on days 0, 3, 6, 9, 13, 16, 18 and 20 of pregnancy.

Food consumption
Food consumption was determined on days 0-3, 3-6, 6-9, 9-13, 13-16, 16-18 and 18-20 of pregnancy.

Mortality and clinical observations
All animals were examined before the start of the study and were shown to be in good general health condition.
The behavior and general health condition of the animals were examined at least twice daily (on weekends and public holidays once daily).

CAESAREAN SECTION AND POST MORTEM EXAMINATIONS
The animals were killed on day 20 of pregnancy and the fetuses removed by Caesarean section.
All animals were examined externally and internally (thoracic and abdominal contents) for macroscopically visible changes, with emphasis on the uterus. Gravid uterus weight was recorded.

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

All animals were examined externally and internally (thoracic and abdominal contents) for macroscopically visible changes, with emphasis on the uterus. Gravid uterus weight was recorded.
The live and dead fetuses in the uterus as well as the conceptuses undergoing resorption and corpora lutea were counted, identified in numerical sequence from cervix to ovary and examined macroscopically. The implantation sites in the uterus were counted after staining with ammonium
sulphide.
The fetuses, the placentae and conceptuses undergoing resorption were removed from the uterus, weighed or measured and examined for gross external abnormalities.

Fetal examinations:

The live and dead fetuses in the uterus as well as the conceptuses undergoing resorption and corpora lutea were counted, identified in numerical sequence from cervix to ovary and examined macroscopically. The implantation sites in the uterus were counted after staining with ammonium
sulphide.
The fetuses, the placentae and conceptuses undergoing resorption were removed from the uterus, weighed or measured and examined for gross external abnormalities. Then the fetuses were killed by CO2 asphyxia.
Approximately 50% of the fetuses of each litter and the dead fetuses were fixed in alcohol, necropsied, sexed and checked for anomalies of the internal organs. The carcasses were placed in a solution of potassium hydroxide for clearing and stained with alizarin red S and Alcian blue. The skeletons were examined and checked for stage of development and abnormalities with the aid of a stereomicroscope.
The remaining fetuses were transferred in Bouin's solution, examined for organ anomalies referring to Wilson's slicing technique [Wilson, J.G.: Embryological considerations in teratology. In Teratology: Principles and Techniques (J.G. Wilson, J. Warkany, Ed.), page 251-277. University of Chicago Press, Chicago, IL (1965)] and sexed.
Visceral and skeletal changes were subdivided into four categories (major defects, minor defects, variations and retardations) based on the severity and/or the spontaneous incidence of the finding.

Statistics:

see below

Indices:

- Pre-implantation loss: Number of corpora lutea - Number of implantations x 100 / Number of corpora lutea
- Post-implantation loss: Number of implantations - Number of live fetuses - x 100 /Number of implantations

FETUSES
- Sex distribution: Number of Male (Female) fetuses x 100 / Number of fetuses
- External abnormalities/litter: Number of fetuses with abnormality x 100 / Number of fetuses
- Visceral abnormalities/litter: Number of fetuses with abnormality - x 100 / Number of fetuses
- Skeletal abnormalities/litter: Number of fetuses with abnormality x 100 / Number of fetuses

Historical control data:

yes

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Body weight development
Mean body weight of all pregnant high dose females (1000 mg/kg bw) was slightly decreased (ca. 5%) against the control, attaining statistical significance (p<0.05) only during late pregnancy (study day 20). Accordingly, overall body weight gain during treatment (study days 6 — 20) was slightly reduced in high dose females (96.9g cf./106.9g for controls). Mean body weight and body weight gains were not influenced by the administration of the test substance in mid and low dose groups.

Food consumption
A transient reduction in absolute food consumption was noted for high dose dams only during the initial treatment period between study days 6-9, with subsequent food uptake comparable to the controls, thereafter. As an incidental finding, relative food intake was slightly significantly reduced against the control during study days 3-6 (prior to treatment). Absolute / relative food consumption was not influenced by the administration of the test substance in mid and low dose groups.

Mortality and clinical observations
Neither deaths nor substance-related adverse clinical signs of intoxication occurred throughout the study. All high dose dams exhibited discolored feces.

Necropsy findings
No substance-related adverse findings were observed at necropsy in all dose groups. Bioavailability of the test substance was confirmed in the kidneys of all dose groups, which were black discolored (all high dose dams) or dark brown (22/23 mid dose dams, 2/23 low dose dams). In addition, the right kidney of one high dose female was enlarged, with a dilated pelvis a second animal of the high dose group showed a with fluid filled kidney. This finding is not unusual for this rat strain and hence, was considered not to be substance-related.

Gestation and caesarean section data
Pregnancy rates accounted 18/23, 20/23, 17/23 and 17/23 females from control, low, mid and high dose groups, respectively. All of these pregnant animals had live fetuses at term. There were no dams with either abortions, premature delivery, or females at term with intrauterine deaths only, at any group.
Early intrauterine deaths accounted 7, 16, 11, and 23 (control — high dose, respectively). Post-implantation loss [%] accounted 3.50, 5.65, 6.41, and 12.82 (control — high dose, respectively), and hence, increased with dose. All percentage figures were within the range of biological variation (<17%) for this rat strain and hence considered not to be biologically relevant. However, a substance-related influence cannot be excluded with certainty although the conceptuses undergoing resorption generally had diameters between 2 and 8 mm with no difference among the groups. There was one case of 16 mm in the control, and 2 cases of 10 mm in the high dose group.
Live fetuses accounted 203, 249, 203, and 169 for the control - high dose groups, respectively, with mean litter sizes of 11.3, 12.5, 11.9, and 9.9, respectively. The lower tendency of live fetuses/litter size for the high dose group against all other groups was not statistically significant, and within the physiological range for this rat strain and age. Hence, it was considered to be possibly incidental, not related to the administration of the test substance.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Findings at caesarean section
There were no findings at caesarian section in any group fetuses, which could be related to the test substance administration.

External, skeletal and visceral examination
Statistical Evaluation (Jackknife t-test, p<0.05) of extemal, visceral or skeletal findings did not reveal any substance-related fmdings in the fetuses of any treatment group.

Findings in dead fetuses
External examination of the dead fetuses did not reveal any substance-related effect.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Only slightly higher post-implantation loss as a secondary effect due to rat specific dental fluorosis in dams resulting from the high amount of a fluoride impurity were observed at the highest dose level

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Slightly lower body weight gains and a slight initial reduction in feed consumption resulting in slightly lower numbers of live foetuses at birth (not statistically significant) and a slightly higher post-implantation loss were observed at at 1000 mg/kg body weight/day. These effects were due to dental problems, resulting from higher fluoride uptake leading to a higher brittleness of the rat teeth. As the registered substance does not containf fluorine or fluoride as an impurity, these effects are of not relevance for the risk assessment of the test substance. Individual examination and statistical evaluation of external, visceral and skeletal effects did not reveal any substance related findings, including the high dose group. Hence the test substance was not teratogenic in the rat at either dose level.
Neither maternal nor embryo-foetal toxicity was observed after administration of at the daily dose of 250 mg/kg body weight. With regard to the present study the 'No Observed Effect Level' (NOEL) is 250 mg/kg bw/day for maternal toxicity and embryotoxicity and the NOAEL is considered to be 1000 mg/kg bw/day.

Executive summary:

The present study was conducted in order to determine the effects of the Structural Analogue 01 (SA01) on maternal state of health, embryonic and foetal development when administered oral by gavage once daily to mated female Sprague Dawley rats from day 6 - 19 of pregnancy.

Groups of 23 mated female Sprague Dawley rats received the test item as a solution in deionized water oral by gavage once daily at the dose levels of 62.5, 250 or 1000 mg/kg body weight from day 6 - 19 of pregnancy (day 0: day of sperm detection) and were sacrificed on day 20 of pregnancy. The dosing volume was 5 mL/kg, corresponding to concentrations of 0, 12.5, 50 and 200 g/L.

Animals were observed daily for mortality and clinical signs of toxicity. Body weight and food consumption were determined regularly throughout the study.

At necropsy the dams were examined for macroscopically visible changes. The uterus was opened and the number of live and dead foetuses and the number of conceptuses undergoing resorption were determined. Body weights, crown-rump lengths, sex ratios of the foetuses and placental weights were determined and external, visceral and skeletal examinations of the foetuses performed.

RESULTS

Neither deaths nor substance-related clinical signs of intoxication occurred throughout the study. The faeces of high dose dams was dark discoloured.

Body weights gains of all pregnant females were slightly decreased in the high dose group (1000 mg/kg bw/day) during the treatment period, attaining statistical significance (p<0.05) at the end of treatment only. Statistical evaluation showed an initial reduction (p<0.05) of feed consumption in this group (study days 3-6) with subsequent recovery thereafter. Effects on body weight and food consumption in the high dose groups were further investigated in the extended one-generation study and were unequivocally related to the dental problems due to the higher fluoride content of the test item. As the target substance does not contain fluoride or fluorine, this effect is not relevant for risk assessment.

Body weight gains and food consumption remained unaffected by the administration of the test substance in the other groups.

 

No substance-related adverse effects were observed at necropsy. The kidneys of all high dose group females, and of a few mid dose group females, were dark discoloured, indicating systemic bioavailability and metabolic burden of the test substance in the body.

There was a slight increase in the number of early or late conceptuses undergoing resorption (post-implantation loss), as well as a slight and not statistically significant decrease in the number of live foetuses at birth in the high dose dams, resulting from the lower body weights and food consumption due to dental problems relating to the higher fluoride intake in this dose-group. No such findings were observed for mid or low dose group females. Foetal crown-rump lengths, litter size, sex ratios, foetal body weight and placental weights remained unaffected by the administration of the test substance in any group. External, visceral and skeletal examinations of the foetuses did not reveal any substance related alterations in any group.

 

CONCLUSION

In conclusion, slightly lower body weight gains and a slight initial reduction in feed consumption were observed in pregnant female Sprague Dawley rats after administration of the test item at the daily dose of 1000 mg/kg body weight/day during days 6-19 of gestation. This was related to dental problems, as the higher fluoride uptake (0.3% fluoride was identified as an impurity of the test substance) leads to a higherbrittleness of the teeth for rats. Hence this effect is not of importance for the target substance. Resulting from the lower feed intake, high dose dams had slightly lower numbers of live foetuses at birth (not statistically significant) and a slightly higher post-implantation loss when compared to the control. In the presence of a normal intrauterine development of the remaining conceptuses, these findings were still on the lower/upper range of biological variation for this rat strain and hence, considered to be of equivocal toxicological significance. Moreover, individual examination and statistical evaluation of external, visceral and skeletal effects did not reveal any substance related findings, including the high dose group.

Neither maternal nor embryo-foetal toxicity was observed after administration of at the daily dose of 250 mg/kg body weight. The test substance was not teratogenic in the rat. With regard to the present study the 'No Observed Effect Level' (NOEL) is 250 mg/kg bw/day for maternal toxicity and embryotoxicity and the NOAEL is considered to be 1000 mg/kg bw/day with regard to human risk assessment.

There was no evidence of developmental toxicity in rats for the test substance, according to the classification criteria of Commission Directive 2001/59/EC.