5-bromo-5-nitro-1,3-dioxane

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 12, 1981

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

5-bromo-5-nitro-1,3-dioxane

EC Number:

250-001-7

EC Name:

5-bromo-5-nitro-1,3-dioxane

Cas Number:

30007-47-7

Molecular formula:

C4H6BrNO4

IUPAC Name:

5-bromo-5-nitro-1,3-dioxane

Details on test material:

- Chemical name: 5-Brom-5-nitro- 1,3-dioxan
- Trade name: Bronidox

Specific details on test material used for the study:

Batch 11 38 232
Stored in glass at room temperature
protected from light
white crystalline powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River WIGA GmbH, Sulzfeld (D).
- Age at study initiation: not given
- Weight at study initiation: 200-240 g
- Fasting period before study: N/A
- Housing: Makrolon cage type 3, Litter: softwood granulate, autoclaved,
- Diet (e.g. ad libitum): Pelleted, low-germ, low-nitrosamine diet Altromin N-1324, Lot 1450, Altromin GmbH, 4937 Lage (D), ad libitum.
- Water (e.g. ad libitum): municipal tap water ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 - 23.5
- Humidity (%): 54
- Air changes (per hr): not given
- Photoperiod (hrs dark / hrs light): 12-hour artificial lighting / day, illuminance
30 - 550 Lux, depending on location.


IN-LIFE DATES: From: 14/12/1983 To: 09/02/1984

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Number and frequence or application:
10 applications in 24 hours intervals from day 6 to day 15 or
gestation. Group 4 : 3 applications only because of the lethal
effects

PREPARATION OF DOSING SOLUTIONS:

The solutions were freshly prepared each day. For applications over the weekend preparations were used, which were scheduled Fridays around 15 clock. The stability of the test substance in the solution was checked.

solutions prepared at 0.05, 0.15 and 0.45 % w/v to be dosed at 10ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical upper test of the test concentrations was carried out 3 times
during the study by photometric determination of Bronidox K in
aqueous solution at 203 nm. For this purpose, 250 μl of each application solution were used
diluted to 25 ml in the flask and the extinctions are determined.

Details on mating procedure:

Mating, the female rats were used in groups of 3 animals / Makrolon cage type 3, the male rats in groups of 4 animals / Makrolon cage type 3.
At the beginning of the mating, 3 female and 1 male animals were assembled. After being diagnosed, the female animals were housed individually in Makrolon cage type 3. The animals were transplanted twice a week.
- Pairing:
Sexually mature females and females were housed in proportion 3: 1 from Monday 8.00 am to Friday 8.00 am.
The insemination was checked by daily checking of the vaginal smear and the vaginal plug. The timing of the appearance of sperm in the vaginal swab or finding the
Vaginal plug was taken as day 0 of pregnancy.

Duration of treatment / exposure:

10 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

15 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

30 (thirty)

Control animals:

yes, concurrent vehicle

Details on study design:

Choice of dosages and the type of application
The highest dose represents about 1/10 of the LD50 value for rats,
the dose of 100/200 mg / kg / day was requested after oral administration
proved to be cumulatively toxic in the long-term trial.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 6, 15 and 19


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: ovaries and uterus

OTHER:

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
- Other: - Number and positions of the implants divided into:
- live fetuses
- dead fetuses early in the uterus
- early-late intrauterine dead fetuses
- Intrauterine dead fetuses
- Total weight uterus with fetuses
- Weight of the placentas

Fetal examinations:

- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [all per litter ]
- Skeletal examinations: Yes: [all per litter ]
- Head examinations: Yes: [all per litter]

Statistics:

student's t-test for body weights and Wilcoxon U-test,
Mann and Whitney for late resorptions

Indices:

Implantations possible:
[Number of implants/Number of corpora lutea] x100

Fetal viability:
[Number of living fetuses/Number of implants] x 100


% of losses before implantation:
{[Number of corpora lutea - number of implants]/Number of corpora lutea} x 100

% of losses after implantation:
{[Number of implants - number of living fetuses]/ Number of implants}x 100


% Sex distribution coefficient:
Number of male fetuses/ Number of fetuses x 100


Number of female fetuses / Number of fetuses x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

From the second application the first symptoms of poisoning appeared.
The intensity of the symptoms as well as the number of affected animals was
dose-dependent. In group 4 (45 mg/kg bw/day) treatment was discontinued after 3 applications for severe general and nervous symptoms.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

1 animal died in the 15 mg/kgbw/day group and 4 died in the 45 mg/kg bw/day group. the dosing of the high dose group was suspended in extremis following the 3rd dose

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

significant decreases in bodyweight gain (P<0.05) in the high dose group on Gestation days 15 and 19

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Group 2: Occasional vocalizations, abnormal gait, vocalization when touched,
Group 3: Isolated vocalizations, vocalization when touched, reduced activity, eg. T. prone position, aggressiveness,
Group 4: Isolated vocalizations, Increased/decreased activity, abnormal gait, decreased reflexes, vocalization when touched, aggressiveness,

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Maternal toxicity is manifested by increased levels of implantation losses (33 in total) at the highest dose level, with 15 and 5 resorptions/implantaiton losses at the low and mid doses respectively

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no total litter losses by resorption

Early or late resorptions:

not specified

Description (incidence and severity):

The timing of the observed resorptions was not specified

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

Maternal toxicity is manifested by increasing numbers of dead fetuses at the highest dose levels compared to other doses administered.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

4 maternal animals died in the high dose group and the remaining 26 animals exhibited symptoms of toxicity. One animal died in the mid-dose groups and 7 other exhibited signs of toxicity.

Effect levels (maternal animals)

Key result

Dose descriptor:

LOAEL

Effect level:

ca. 5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

clinical signs

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

not examined

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

offspring viability reduced to 87.73% compared to 97.63% in control ( not statistically significnat)

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

one instance of ex-encephaly and two instances of thoracic spina bifida, including one with absence of lumbar and sacral vertebra in mid-dose group

Visceral malformations:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 15 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

Fetal abnormalities

Abnormalities:

effects observed, non-treatment-related

Localisation:

skeletal: skull

skeletal: sternum

skeletal: vertebra

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

45 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

Based on the study described Bronidox K is to be classified as maternally cumulatively toxic, non-embryolethal as well as non-teratogenic. Anyfetal effects (resorptions/fetal deaths) observed are a direct result of non-specific secondary consequence of maternal toxicity.

Executive summary:

Bronidox K was applied orally in doses of 5, 15 and 45 mg/kg body weight/day once daily to groups of pregnant rats from day 6 to day 15 of pregnancy. Control animals were treated simltaneously with the vehicle. All dosages resulted in general symptoms of intoxication with one death in the median dose group of 15 mg/kg/day, and 4 animals died after 2 to 3 applications of the highest dose of 45 mg/kg/day. An influence of the test substance on fetal parameters was recognisable only at the highest dosage by an increased resorption rate after implantation and by increased rate of retardation. The investigation of the foetuses yielded no indication of direct embryotoxic or teratogenic effects. Based on the study subsequently described Bronidox K is to be classified as maternally cumulatively toxic, non-embryolethal as well as non-teratogenic.