2,2'-iminobis[4,6-diamino-1,3,5-triazine]

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April - August 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study with GLP. The English translation of the Chinese report sometimes speaks of a "28-day study" or similar mixed up text with the also available 28-day study. By comparing with the Chinese text, where the numbers are given in arabic figures, these errors of the translation could be cleared.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: "The Guidelines for the Testing of Chemicals 2004" No. 408, China.

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: HJ/T154-2004, "The Guidelines for the Hazard Evaluation of New Chemical Substances"

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

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:

N~2~-(4,6-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
Sample characters: off-white powder
Physicochemical property: /
Storage: At room temperature, ventilated and dry

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shanghai Experimental Animal Feed Center. Animal Yield Certification: SCXK (Hu) 2008-0016.
- Age at study initiation: 6-8 week-old
- Weight at study initiation: male rats: 182- 218g, female rats: 181 - 219g
- Fasting period before study:
- Housing: suspended cages (L46 xW31 xH20cm). Rats of experimental groups were individually housed while 5 rats of satellite groups were housed in one cage.
- Diet: ad libitum, laboratory diet was provided by Suzhou Shuangshi Laboratory Animal Feed Science and Technology LTD.
- Water: ad libitum, Drinking water was conducted in compliance with GB5749 "Hygienic standard for drinking water"
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +3°C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

0 mg, 188 mg, 750 mg and 3000 mg of test substance were weighed respectively, and suspended evenly with 30mL of 1 % carboxymethyl cellulose to prepare as the vehicle control, low dose, middle dose and high dose groups.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Due to the poor solubility of the test substance, and the results of acute oral toxicity test, 1% carboxymethyl cellulose was chosen as the solvent.
- Amount of vehicle (if gavage): 30 mL

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

The rats were exposed with the test substance five days a week for a period of 90 days.
The gavage volume was 1 ml/100 g body weight (the sample solution was suspended evenly while administrating).

Frequency of treatment:

five days a week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg BW
Basis:

Doses / concentrations 2

Remarks:

Doses / Concentrations:
62.5 mg/kg BW
Basis:

Doses / concentrations 3

Remarks:

Doses / Concentrations:
250 mg/kg BW
Basis:

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1000 mg/kg BW
Basis:

No. of animals per sex per dose:

20 for each group (10 males and 10 females)

Control animals:

yes, concurrent vehicle

Details on study design:

Two additional satellite groups, dosed with 0 mg/kg respectively 1000 mg/kg bw were also employed. Each of the satellite groups had 5 m + 5 f animals.
Animals in satellite groups scheduled for follow-up observations were housed for a further 14 days without treatment in order to detect delayed occurrence of persistence or recovery from toxic effects once a day.

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day for 90 days.

BODY WEIGHT: Yes
- Time schedule for examinations: weighed before exposure and. once a week after administration

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 24 hours after final administration.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals:
- Parameters checked in table 10 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 24 hours after final administration.
- Animals fasted: No data
- How many animals:
- Parameters checked in table 11 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: last week of the study using timed urine volume collection of 24h.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Several parameters were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes ;
The liver, kidney, adrenals, uterus, oviduct, testes, lung, spleen, brain and heart of all
animals were weighed and the weight coefficiency of each organ was calculated.
A full, detailed gross necropsy which included careful examination of the body was subjected
to all animals died during and after administration. All gross lesions were examined and main
organs were preserved in 10% neutral buffered formalin.
HISTOPATHOLOGY: Yes;
Full histopathology was carried out on the preserved brain, pituitary gland, stomach, small
and large intestines, liver, kidney, adrenals, spleen, pancreas, heart, thyroid gland, parathyroid
gland, trachea and lungs, uterus, prostate, ovary, testes, urinary bladder, lymph nodes and aorta
of all animals in the control and high dose groups. These examinations will be extended to
animals of middle and low dose groups, if any treatment-related changes are observed in the
high dose group.

Statistics:

All data and results were summarized and analysed with SPSS 13.0 to calculate the No Observed Adverse Effects Level (NOAEL) of the rats.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During exposure, some animals in high dose groups presented abnormal pattern of breathing. After stopping exposure, this toxic sign was gradually recovered. Four animals of the high dose group showed emaciation from day 7 of gestation on.

Mortality:

mortality observed, treatment-related

Description (incidence):

During exposure, some animals in high dose groups presented abnormal pattern of breathing. After stopping exposure, this toxic sign was gradually recovered. Four animals of the high dose group showed emaciation from day 7 of gestation on.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
During exposure, some animals in high dose groups presented abnormal pattern of breathing. After stopping exposure, this toxic sign was gradually recovered.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

No signs of intoxication were observed in both male and female rats.
No Observed Adverse Effect Level (NOAEL) of male and female rats were both 1000mg/kg.

Executive summary:

Guideline: This study of 2,2'-iminobis[4,6-diamino-1,3,5-triazine] (CAS No.:3576-88-3) was conducted in compliance with "The (Chinese) Guidelines for the Testing of Chemicals 2004" No. 408.

Animals: 120 healthy Sprague-Dawley rats, 60 male and 60 female, body weight range of male rats was 182-218 g, and that of female rats was 181-219 g.

Method: The test item 2,2'-iminobis[4,6-diarnino-1,3,5-triazine] (CAS No.:3576-88-3) was administered to male and female SD rats at concentrations of 0, 62.5, 250, and 1000mg/kg body weight respectively. Two additional satellite groups were set up in 0 mg/kg and 1000mg/kg. Each rat was weighed and randomly assigned to the control, experimental and satellite groups (10 per sex). The animals in the control and experimental groups were housed individually, whereas animals of the same sex in the satellite groups were caged in small groups of 5. The rats were dosed with the test item daily five days each week for a period of 90 days.

After treatment, all animals were observed and recorded for morbidity and mortality.

All animals were weighed once a week and the measurement of food consumption was also made once a week.

The 24h urine sample of each rat was collected and analysed in the fourth week of the study.

Haematological examinations and biochemical examinations were performed on rats' blood samples, which were obtained 24 hours after final dosing.

At the end of this study, all animals were executed and subjected to a full gross necropsy.

The liver, kidney, adrenals, uterus, salpinx, testes, lung, spleen, brain and heart of all animals were weighed and weight coefficient of the organs was calculated.

Brain, pituitary gland, stomach, small and large intestines, liver, kidney, adrenals, spleen, pancreas, heart, thyroid gland, parathyroid gland, trachea and lungs, uterus, prostate, ovary, testes, urinary bladder, lymph nodes and aorta of all animals in the control and in the high dose groups were performed histopathological examinations.

Animals in satellite groups scheduled for follow-up observations were kept for a further 14 days without treatment to detect possible delayed occurrence, of persistence or recovery from toxic effect.

All data and results were summarized to demonstrate the no observed adverse effects at the lowest dose level (NOAEL) of the test animals.

Conclusion: There is no obvious toxic effect of animals in 90-days repeated dose toxicity test. No

observed adverse effect at the lowest dose level (NOAEL) of rats for each sex was 1000 mg/kg.