5,5'-Bi-2H-tetrazole, ammonium salt (1:2)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

3 (not reliable)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

485-290-0

EC Name:

-

Cas Number:

3021-02-1

Molecular formula:

C2H8N10

IUPAC Name:

5-(1H-1,2,3,4-tetrazol-5-yl)-1H-1,2,3,4-tetrazole diamine

Constituent 2

Reference substance name:

5,5'-Bi-1H-tetrazole diammonium salt

IUPAC Name:

5,5'-Bi-1H-tetrazole diammonium salt

Test animals

Species:

rat

Strain:

Wistar

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Preparation of test item:
1. Required amount of BHT-2NH3 5,5 ́-Bis-1H-tetrazole diammonium salt was weighted into an appropriate vial on an analysis scale
2. Water was added up to the required volume and the liquid was stirred for at least 5 minutes
3. Liquid was stirred briefly immediately before drawing it into the gavage

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Concentration was not verified.

Details on mating procedure:

The animals were delieverd from the supplier still being gravid. The pregnancy status was concluded by finding of a mating plug and vaginal smears. The day of sperm plug detection was designated as the Day 0 of gestation.

Duration of treatment / exposure:

from Day 6 until Day 20 of gestation

Frequency of treatment:

daily

Duration of test:

14 d

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
250 mg/kg bw
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50 mg/kg bw
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
10 mg/kg bw
Basis:
nominal conc.

Doses / concentrations 4

Remarks:

Doses / Concentrations:
0 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

Background of mating procedure and pregnancy status:

The pregnancy status was concluded by finding of a mating plug and vaginal smears. The day of sperm plug detection was designated as the Day 0 of gestation.
The test animals were ordered to arrive at the test facility at two separate dates two weeks apart, each shipment containing two cohorts of 25 animals each - one cohort at Day 0 and the other cohort at Day 1 of gestation. Although 100 gravid female rats were ordered from the animal supplier in four cohorts of 25 animals each, the actual numbers of gravid females depended on the mating success of test animals at the animal supplier.
Rationale for the dose levels:


Rationale for the dosage:
In a subacute toxicity study according to OECD 407, the administration of the test item at a dose level of 1000 mg/kg over 28 days produced mild but not toxic effects in non-gravid Wistar rats2. (raised food and water consumption, urea nitrogen level and leukocyte counts and lowered body mass in both sexes at the high dose group, )
In a dose range finding (DRF) study, the test item was administered to 15 gravid Wistar WU rats at three dose levels (1000 / 500 / 250 mg/kg body weight) from Day 5 to Day 20 of gestation.
Within the parameters monitored in that assay, oral uptake of the test item at three dose levels of 250, 500 and 1000 mg/kg body weight over a treatment period of 15 days did lead to strong effects in gravid Wistar rats of the high dose group and the medium dose group. Effects of the test item were mild but considerable in the low dose group. Since medium and high dose of the test item seemed to have an effect on the gravidity of treated females, dosage of the main toxicity study should not exceed 250 mg/kg body weight. Taking these results into consideration 250 mg/kg was determined as high dose for the present prenatal developmental toxicity study.

Examinations

Maternal examinations:

The following parameters were observed and documented during the in-life period:
- Viability / fatalities Daily
- General clinical signs / behaviour: Daily
- Detailed clinical signs: Daily
- Body mass: Days 1, 6, 9, 12, 15, 18, and 21
- Food consumption: Days 1, 6, 9, 12, 15, 18, and 21
- Water consumption, Days 1, 6, 9, 12, 15, 18
- Viability, general clinical signs and behaviour
- Fatalities
- changes in skin, fur, eyes, mucous membranes
- occurrence of secretions and excretions
- autonomic activity (e.g. lacrimation, piloerection, unusual respiratory patterns)
- changes in gait, posture, and response to handling
- presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards)
- and any other observed, unusual signs.

Exminatiosn after necroscopy:
-Examination of uterine content
-Examination of foetuses

Ovaries and uterine content:

Examined were:
Uterus mass
Number of Corpora Lutea and Implantations
Number of viable foetuses and degree of resorptions

Fetal examinations:

The sex and body weight of each viable foetus as well as the weight of the respective placenta was determined and each foetus was examined for external alterations at the test facility. One-half of each litter was prepared and stained at the test facility to investigate skeletal alterations (variations and malformations), which were examined at the PI. The remainder foetuses were fixed in modified Davidson solution at the test facility and subsequently prepared and examined at the PI for soft tissue alterations (variations and malformations), using serial sectioning methods

The foetuses had to be re-fixated at the Principal Investigator for at least 5 days using Davidson solution containing 10% formalin as originally stated in SOP RDP/R2 07.03 of the PI, since fixation of Wilson foetuses at the test facility was performed using a formalin content of 4% instead of 10% by error. Therefore, the start of foetal evaluation of the remaining Wilson foetuses had to be postponed from CW 16 to CW 18 2009. In order to minimise the risk of total data loss due to possible staining artefacts, the staining of Dawson foetuses was performed subsequently in cohorts of 10-30 litters. As a result, shipment of foetuses and the start of evaluation at the PI were delayed as stated above.

Indices:

Calculated indices:
Pre implantation loss: mean no. of corpora lutea – mean no. of implantations
Mean no. of resorptions: No. of resorptions / no. of litters
Mean pre implantation loss per group: (mean no. of corpora lutea – mean no. of implantations) * 100 / mean no. of implantations
Mean post implantation loss (%) = (Total no. resorptions + total no. of dead foetuses) * 100 no. of foetuses. All calculations and statistical tests were documented within the appendix.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Markedly reduction of body mass gain, in combination with reduction of food consumption and moderate increase of water consumption in the highdose group.
Mean uterus mass was markedly reduced in the high dose group.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

>= 0 - < 10 mg/kg bw/day (nominal)

Basis for effect level:

other: developmental toxicity

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Teratogenic effects occurred in form of malformations of the eye (anophthalmia, aphakia, retina folds, and lens alterations) at all three dose levels (10, 50 and 250 mg/kg/d) and in form of malformations of the great blood vessels (absent innominate artery and right sided aortic arcli) at the medium and high dose levels (50 and 250 mg/kg/d). Incompletely ossified bones and variations of the ribs (thickened or wavy ribs) were additionally seen in all three dose groups (10, 50 and 250 mg/kg/d) with the highest incidence in the high dose group (250 mg/kg/d).

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Toxiceffects:

Dams
Parameter	Vehicle	Low dose	Medium dose	High dose
Dams per cohort	25	25	25	25
Successful mating	23	23	24	21
Fatalities	None	None	None	None
Viability, clinical signs andbehaviour	NAD	NAD	NAD	NAD
Detailed clinical signs	NAD	NAD	NAD	NAD
Body mass	NAD	Mildly altered	Mildly altered	Reduced
Food consumption	NAD	NAD	Raised	Raised
Water consumption	NAD	NAD	NAD	Raised
Necropsy	NAD	NAD	NAD	NAD
Uterus mass	NAD	NAD	Mildly raised	Reduced
Mean pre implantation losses / group (%)	NAD	NAD	NAD	NAD
Mean post implantation losses / group (%)	NAD	NAD	NAD	Raised
Mean No.ofresorptions	NAD	NAD	NAD	NAD
Mean no.offoetuses	NAD	NAD	NAD	NAD
Foetuses	Vehicle	Low dose	Medium dose	High dose
Meanfoetalmass	NAD	NAD	Females reduced	Reduced
Gender biasfoetusmass	NAD	NAD	NAD	NAD
Mean placental mass	NAD	NAD	Males reduced	Reduced
Incidence ofteratologicalmalformations at visceral examination* No offoetuses examined (no.oflitters) EyeBlood vessels	Yes n=111 (23) NAD 1	Yes n=111 (23) 5 NAD	Yes n=119 (24) 10 3	Yes n=100 (21) 9 5
Incidence ofteratologicalmalformations at skeletal examination* No offoetuses examined (no.oflitters) Thickened ribs Wavy ribs	NAD n=125 (23) NADNAD	Yes n=122 (23) 7 4	Yes n=133 (24) 25 13	Yes n=109 (21) 97 107

NAD = No abnormality detected

*Teratogenic effects occurred in form of malformations of the eye (anophthalmia,aphakia, retina folds, and lens alterations) at all three dose levels (10, 50 and 250 mg/kg/d) and in form of malformations of the great blood vessels (absent innominate artery and right sided aorticarcli) at the medium and high dose levels (50 and 250 mg/kg/d). Incompletely ossified bones and variations of the ribs (thickened or wavy ribs) were additionally seen in allthreedosegroups (10, 50 and 250 mg/kg/d) with the highest incidence in the high dose group (250mg/kg/d).

Applicant's summary and conclusion

Conclusions:

Teratogenic effects occurred in form of malformations of the eye (anophthalmia, aphakia, retina folds, and lens alterations) at all three dose levels (10, 50 and 250 mg/kg/d) and in form of malformations of the great blood vessels (absent innominate artery and right sided aortic arcli) at the medium and high dose levels (50 and 250 mg/kg/d). Incompletely ossified bones and variations of the ribs (thickened or wavy ribs) were additionally seen in all three dose groups (10, 50 and 250 mg/kg/d) with the highest incidence in the high dose group (250 mg/kg/d). The effects on the ribs were clearly dose-dependent. Therefore, the NOAEL for embryo-foetal development was below the low dose of 10 mg/kg/d due to substance-related effects on the eye, the great blood vessels, and ribs.