Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The center has been assessed by China National Accreditation Services for Conformity Assessment (CNAS) and proved to be in compliance with CNAS/CL01:2006 'Accreditation Criteria for Testing and Calibration Laboratories (Identical to ISO/IEC17025: 2005 General Requirements for the competence of Testing Calibration Laboratories). The accreditation certificate number is CNAS L0238.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
not specified
Remarks:
China National Accreditation Services for Conformity Assessment (CNAS) and proved to be in compliance with CNAS/CL01:2006 'Accreditation Criteria for Testing and Calibration Laboratories

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Sample No: ZL0902374
- Supplied by: Thomas Swan & Co,. Ltd
- Physical appearance: Clear colourless liquid, soluble in water and plant oil
- Storage - test substance was stored in its original container that supplied by Thomas Swan & Co,. Ltd at toom temperature
- Handling - The test substance was handled with necessary protective methods and all recommended safety measures were followed.
-

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
- Supplier: Guangdong Medical Laboratory Animal Center [certified animal number SCXK (Guangdong) 2008-0002].
- Number of animals used: 200 male and 200 female
- Number of groups: 5 groups ( a negative control, a positive control, a low-dose, a medium-dise, a high-dose)
- Number of animal in each group: 145 young adult nulliparous female rats were successfully mated with the males of the same species and strain, and then they were randomly divided into 5 groups (29 mated females in each group).
- Body weight at start of experiment: 200 ~ 290g before mating
- Identification of animals: Tags marked with animal group number and treatment detaisl were attached to cages. Each animal was given a unique number.
- Acclimatisation: 5 days prior to the experiment in the test room
- Randomisation: Animals were randomly assigned to 5 groups based on their body weights.

- Test Room: SPF animal lab in the Center.
- Animal house condition: The SPF test facility was an air-conditioned room with 12 hours artifical fluorescent light and 12 hours dark
- Temp range: 20~25°C
- Humidity Range: 40~70%
- Caging: Stainless steel cages were used at first. when animals were mated, the females were housed in plastic cages in small numbers.
- Water bottle: Each cage was supplied with a polypropylene water bottle with a stainless steel nozzle.
- Sanitation: Sterilised bedding material was changed 3 times a week. Corncob Laboratory Animal Bedding was supplied by Guangzhou Sebiona Bio-Tech Co,. Ltd.
- Food and Water: Standard pellet feed supplied by Guangdong Medical Laboratory Animal Center and ultra-pure filtered sterilized water were provided to the animals freely.
-Freq. of providing feed and drinking water: Both drinking water and feed were provided ad libitum.

Examinations

Ovaries and uterine content:
Immediately after termination or as soon as possible after death, the uteri were removed and the pregnancy status of the animals ascertained. Uteri that appear non-gravid should be further macroscopically examined to confirm the non-pregnant status. Gravid uteri (foetuses attached and not-attacthed) including the cervix were weighed. The number of corpora lutea was counted for the pregnant animals. The uterine contents were examined for numbers of embryonic or foetal deaths and viable foetuses. The degree of resorption was described in order to estimate the relative time of death of the conceptus. Net weight increase of a dam was then calculated (bw on day 20 - bw on day 6 - weight of gravid uteri attached foetuses).
Fetal examinations:
The sex, body weight, body length and tail length of each foetus were determined. Each foetus was examined for external alterations. Then 2/3 of the foetuses were prepared (mainly including transparent and dyeing steps) amd examined for skeletal alterations with dissection microscope. The remainder foetuses were examined for soft tissue alterations (e.g. variations and malformations or anomalies) using appropriate serial sectioning methods and observed with dissection microscope. Categorisation of foetal alterations were listed.
Statistics:
Statistical method:Numerical results were evaluated by statistical method ofF testor x2 test using the litter as the unit for data analysis.

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
25.28 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
Dose descriptor:
NOAEL
Effect level:
126.4 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
Dose descriptor:
NOAEL
Effect level:
632 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs

Results (fetuses)

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
25.28 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

RESULTS

Status of gestation: 29 young adult nulliparous female rats in each group were successfully mated but the actually pregnant numbers in each group were between 23-27 (Table 3). The embryonic resorptions in low-, medium- and high-level groups were significantly increased, while the viable foetuses were decreased (P<0.05 or P<0.01). In medium- and high-dose groups, the foetal deaths were observed to be higher than that of the control(<0.01). Compared with those of the control, the increased embryonic resorption rate, increased foetal deaths and decreased viable foetus rate in the positive group were significantly different (P<0.01). There were no differences between sex ratio of the foetuses in all dose groups.

Foetus growth: Compared with those of the control, the body weight, body length and tail length of the foetuses in the high dose group and in the positive group were significantly decreased (P<0.01)

Foetuses external alterations:Short-tailed, kinked-tailed, acaudate and malrotated-foot foetuses were found in the positive group (P<0.01). Some signs were also found in dose groups but without any signicant difference with the contros(P>0.05)

Foetuses soft tissue alterations:No soft tissue alterations were observed in all dose groups. One case of ventriculomegaly was found in the high dose group but without difference with the controls (P>0.05). Some malformations were found in the positive group(<0.05 or<0.01), showing N,N'-Methylene bis -(2 -amino -1,3,4 -thiadiazole) could induce soft tissue alterations in rats

Foetuses skeletal alterations:Some types of malformations were found in the high dose group, which partly were significantly different with those of the control (^O.OS or<0.01). The skeletal alterations were mainly including enlarged fontanel, abnormal upper occipital morphology, dysostosis of parietal bones and os incae, bipartite ossification of cervical arch and sternum, and abnormal metacarpal ossification (Table 7).

Some malformations were found in the positive group with high incidence(<0.05 or P<0.01), showing N,N'-Methylene bis -(2 -amino -1,3,4 -thiadiazole) could induce skeletal alterations in rats.

Evaluation of Results:

(1) Maternal toxic response: No obvious maternal toxic response was observed under designed dose levels.

(2) Embryonic toxic response: Definite embryonic toxic responses were observed at the low-, medium- and high-dose groups under the existing test conditions.

(3) Teratogenicity: Under the existing test conditions, 632.0mg/kgethyl tetrahydrofurfuryl etherelicited the increase of skeletal malformations. The minimum teratogenic dose was 632.0mg/kg and theNo Observed Adverse Effect Level (NOAEL)ofethyl tetrahydrofurfuryl ether(supplied by Thomas Swan&Co. Ltd.) was 126.4mg/kg on SPF SD rats. Based onThe Guidelines for the Testing of Chemicals (No. 414’ Teratogenicity)of the State Environmental Protection Administration of China, theTeratogenicity Indexwas calculated to be 5 (3160mg/kg/632.0mg/kg), indicating this test material was categorized as "Basically Non-teratogenic".


Applicant's summary and conclusion

Conclusions:
The minimum teratogenic dose was 632.0mg/kg and the No Observed Adverse Effect Level (NOAEL) of theyl tetrahydrofurfuryl ether (supplied by Thomas Swan & Co. ltd) was 126.4mg/kg on SPF SD rats.