Registration Dossier

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:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

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 / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
250 mg/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
50 mg/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
10 mg/kg bw
Basis:
nominal conc.
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.