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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:
March - August 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: The Guidelines for the Testing of Chemicals (2004) 414, China.
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
other: HJ/T154-2004, "The Guidelines for the Hazard Evaluation of New Test Chemical Substances", China.
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-iminobis[4,6-diamino-1,3,5-triazine]
EC Number:
222-695-1
EC Name:
2,2'-iminobis[4,6-diamino-1,3,5-triazine]
Cas Number:
3576-88-3
Molecular formula:
C6H9N11
IUPAC Name:
2-N-(diamino-1,3,5-triazin-2-yl)-1,3,5-triazine-2,4,6-triamine
Details on test material:
Sample name: 2,2'-iminobis[4,6-diamino-1,3,5-triazine]
CAS No.:3576-88-3
Supplier: DSM Engineering Plastics.
Physical state: off-white powder
Sample storage: At normal room temperature and humidity in the lightproof sample storeroom

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: SHANGHAI SLAC LABORATORY ANIMAL CO. LTD
- Age at study initiation: 12 weeks old
- Weight at study initiation: males: 300-400g and females: 237-328g.
- Housing: Animals were housed individually in plastic cages (L46 xW31 xH20cm).
- Diet (ad libitum): Diet were provided by SUZHOU SHUANGSHI LABORATORY ANIMAL FEED SCIENCE CO.,LTD. Certification: Su (E) Sishengzi (2002) 006.
- Water (ad libitum): The quality level of drinking water is in accordance with the standard of national GB5749 (Standards for Drinking Water Quality).
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25 °C
- Humidity (%): 40-70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): alternating 12 h periods of light and darkness

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
0, 250, 500 and 1000 mg of test substance were weighed and dissolved in 1% CMC-Na to a
volume of 10 ml as each solution of negative control, low, middle and high dose groups.
2 mg of Dexon was dissolved in 10 ml distilled water as solution of the positive control group.

VEHICLE
- Amount of vehicle (if gavage): 1 mL/100 g BW
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: One male rat and two female rats
- Length of cohabitation: from the afternoon to the next morning
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
From day 6 to day 15 of gestation
Frequency of treatment:
Daily from day 6 to day 15 of gestation
Duration of test:
19 days (All of female rats were executed one day prior to the expected day of
delivery, which was day 20 of gestation.)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
250 mg/kg BW
Basis:

Remarks:
Doses / Concentrations:
500 mg/kg BW
Basis:

Remarks:
Doses / Concentrations:
1000 mg/kg BW
Basis:

No. of animals per sex per dose:
20 pregnant female rats per dose.
Control animals:
yes, concurrent vehicle
other: positive control: DEXON
Details on study design:
- Dose selection rationale:
Preliminary test before formal trial was performed to determine the high dose in the
teratogenicity test in rats. According to the LD50 (5000mg/kg bw) calculated from the acute
toxicity test and the NOAEL (500mg/kg) obtained from 28 days of short-term repeated toxicity
test, the dose of 1000mg/kg ( 1/5 LD50 ) was first selected and the non-pregnant female rats (n=20)
were gavaged (1ml/100 mg BW) for 10 days. The toxic signs and the death were observed daily till
the 15th day.
Results of preliminary test: Different degree toxic signs were seen and none females died,
which was permitted by the principle of dosage design (toxic reaction is allowed by the high
dose, but the mortality rate should not exceed 10%). Therefore, the high dose of 1000mg/kg (1/5
LD50) was started in formal test.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table 2 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: every 3 days from the day 6 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20

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
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes
Statistics:
All the data were summarized and analyzed by statistical software SPSS13.0. The measured
data, such as fetus body weight, body length, tail length and placenta weight, were tested by
one-way ANOVA (Dunnett tests). The resorption and dead incidence of fetus were analyzed by
Ch2 tests based on the parent body as statistical unit. The enumeration data, such as the resorption
and the dead rate of fetus, the teratogenic rate of skeleton and soft tissue, were compared between
treated groups and the negative control group by the approach of binomial distribution based on
the fetus number as statistical unit. The dose will be regarded as the teratogenic effects if the
difference of total teratism rate or individual teratism rate between such dose group and the
negative control group was statistically significant.
Total teratism rate in each group (%) = the total number of viable fetus with teratism divided by the total
number of viable fetus in parent body of this group x 100 % (One teratism was counted while
more than one kind of malformation happening in the same viable fetus). Teratogenicity index =
LD50 of female animals/the minimum teratogenicity dosage.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Compared with the negative control group, 1000 mg/kg (high dose) of this chemical significantly inhibited the body weight gain of pregnant rats,
suggesting that it had maternal toxicity to a certain extent. The other two doses had no such remarkable effects, suggesting that this test
met the requirement of dosage design principle in teratogenicity study.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
500 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:
The high, middle and low doses of this chemical didn't significantly effect embryogenesis (no induction on the
resorption and death of fetus) and the growth and development of the fetus (body weight, placenta
weight, body length and tail length, sex ratio, the formation of appearance and soft tissue as well as
the development of skeletal system).

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
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

Compared with the negative control group, the positive reagent could inhibit the body weight gain of pregnant rats, inducing growth retardation of pregnant rats. In addition, Dexon could increase the resorption incidence of fetus in pregnant rats, reduce body weight, placenta weight, body length and tail length of fetus and induce the abnormality of appearance, skeleton and soft tissue of fetus obviously, indicating the establishment of the experiment methods in this study.

Applicant's summary and conclusion

Conclusions:
This teratogenicity test in SD rats indicated that the body weight gain of pregnant rat
by 1000mg/kg was reduced significantly, compared with that of the negative control, indicating that
2,2'-iminobis[4,6-diamino-1,3,5-triazine] (CAS No.:3576-88-3) had maternal toxicity to a certain
extent; NOAEL for maternal toxicity is 500 mg/kg.
No embryonic toxicity and teratogenicity were detected in this study. The teratogenic NOAEL was 1000mg/kg.
According to the assessment of HJ/T 154-2004, the result of the teratogenicity test in rats is negative.
Executive summary:

The teratogenicity test of 2,2'-iminobis[4,6-diamino-1,3,5-triazine] in rats was performed according to The Guidelines for the Testing of Chemicals (2004) 414.

Experimental Animals:

Sprague-Dawley (SD) rats: 3 months old, 260 sexual maturity and unmated females, 237-328g

of body weight and 120 males, 300-400g of body weight.

Experimental method:

Three different dose-level test groups of low, middle and high dosage, a negative control

group and a Dexon positive control group were set up in this test. The dosage of each test group

was 250 mg/kg, 500 mg/kg and 1000 mg/kg BW, respectively. 20 pregnant female rats were

assigned to each group. The day on which a vaginal plug or sperm were observed was counted as

day 0 of gestation. From day 6 to day 15 of gestation, 1ml/100g BW of test substance was

administered daily by gavage to rats in 3 different dose groups as well as control groups.

Clinical observations were recorded every day. From day 6 of gestation, rats were weighed every 3 days

during the dosing period.

All of female rats were executed one day prior to the expected day of delivery, which was day 20 of gestation.

Immediately after caesarean, the uteri were removed and gravid uteri were weighed.

Examination of uterine contents was counted: the number of corpora lutea, implantation, resorptions,

viable fetuses and embryonic or fetal deaths, etc.

The development of fetuses was recorded one by one: body weight, body length, tail length, gender etc.

Examination of teratogenicity of fetuses was performed later: their appearance, skeletal and soft tissue alterations.

Statistical software was applied to analyze all the data and results.

The maximum no observed adverse effect level (NOAEL) of teratogenicity test was obtained through statistical analysis.

Result and conclusion:

This teratogenicity test in SD rats indicated that the body weight gain of pregnant rat by 1000 mg/kg reduced significantly, compared with that of the negative control, indicating that 2,2'-iminobis[4,6-diamino-1,3,5-triazine] had maternal toxicity to a certain extent; NOAEL for maternal toxicity was 500 mg/kg.

No embryotoxicity and teratogenicity were detected in this study. The maximum teratogenic NOAEL was 1000 mg/kg. According to the assessment of HJ/T 154-2004, the result of the teratogenicity test in rats is negative.