Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1 March 1978 to 29 February 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study was performed before GLP and OECD guidelines. However the method used is similar to to OECD 414. The restriction is also due to the use of the read across approach: the test was performed not with HMX but with RDX, a substance which has been demonstrated to be very similar in structure, physical/chemical properties and toxicological profile .

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Perhydro-1,3,5-trinitro-1,3,5-triazine

EC Number:

204-500-1

EC Name:

Perhydro-1,3,5-trinitro-1,3,5-triazine

Cas Number:

121-82-4

Molecular formula:

C3H6N6O6

IUPAC Name:

Perhydro-1,3,5-trinitro-1,3,5-triazine

Constituent 2

Reference substance name:

RDX

IUPAC Name:

RDX

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): RDX, heaxhydro-1,3,5-trinitro-1,3,5-triazine
- Molecular formula (if other than submission substance):C3H6N6O6
- Molecular weight (if other than submission substance): 222.26
- Smiles notation (if other than submission substance):N(=O)(=O)N(CN(N(=O)(=O))CN1N(=O)(=O))C1
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type: Explosive
- Physical state: crystal
- Analytical purity: 88.6% +/- 0.9%
- Impurities: 2.2% +/- 0.1% water and 9% HMX
- Lot/batch No.: HOL 43537

Test animals

Species:

rat

Strain:

Fischer 344

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Well known breeder
- Housing: Animal were usually housed together in plastic shoebox cages witb corn cob bedding
- Diet (e.g. ad libitum): ad libitum)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: one week

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% methylcellulose and 1% polysorbate 80 in water

Details on exposure:

Before preparation of the suspension, bulk RDX was crushed by hand. A concentrated suspension was prepared by hand mixing and diluted to proper concentrations with magnetic stirring. The vehicle was 1% methylcellulose and 1% polysorbate 80 (Tween 80, Lot No. 766613 or Lot No. 781666, Atlas Chemical Industries) ("MCTW") in water. Maintaining uniform suspensions was not always easy. Freeze-thaw cycles helped, especially with the more concentrated suspensions.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A variety of dosage preparation were assayed for RDX content. Some of these were trial mixes to develop methods and check stability of the preparation. Others were actual dosage preparations, spot checks, rather than a systematic assay. Sample aliquots were diluted with acetone to give a concentration of ~ 15 mg RDX/mL and assayed by HPLC-UV.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: until evidence of mating
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged : the male was removed from the cohabitation cage

Duration of treatment / exposure:

Rats were dosed by gavage on days 6 through 19 of gestation

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.2 mg/kg/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
2 mg/kg/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
20 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

24 female rats per dose for all treated groups except for the highest dose group which contained 25 female rats

Control animals:

yes, concurrent vehicle

other: Positive control 350mg/kg hydroxyurea given on Day 6 followed by vehicle on Day 7 to Day 12

Details on study design:

- Dose selection rationale: based on a rat range finding study performed by gavage with 10, 20, 40 and 80 mg RDX/kg/day.
All rats given a RDX dose of 80 or 40mg/kg/day died following convulsions. Inspection of the uterus at the time of death indicated that all dams in the 40 mg/kg/day but not in the 80 mg/kg/day dose group had hemorrhages around implantation sites or detached feto-placental units. Similar effects were not observed at lower doses with full-term dams. With the exception of one dam given 20 mg/kg/day which exhibited some of the jerking movements, no effects were seen in dams given 10 or 20 mg/kg/day when compared with dams given the suspension medium without RDX. An indication of morbidity was evident in the weight gain of these dams. Mean values for the treatment weight change (difference between gravid day 20 and day 6 bodyweight) were 91 or 86 g for dams given 10 or 20 mg/kg/day versus 126 g for the control. No indications of an adverse effect on fetal development was observed. This range finding teratology study in rats suggests that at dosage levels of 20 mg/kg/day RDX a consistent adverse effect on maternal weight gain and intrauterine effects may be observed. As a result of this study, three dose levels of RDX (i.e., 0.2, 2, and 20 mg/kg/day) were selected for the full-scale study.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: on gestation days, 6, 7, 9, 11 and 19

FOOD CONSUMPTION : Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- the viscera examined grossly

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: one third of the viable fetuses per litter
- Skeletal examinations: Yes: two third of the viable fetuses per litter

Statistics:

Data were analyzed by a nonparametric rank test. The level of significance was selected a p<0.05 unless otherwis indicated. The litter was considered the experimental unit of observation. For example, the percent of fetuses with a given anomaly was calculated for each litter. These percentages were then analysed by a non parametric rank test.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY:
Mortality was observed in 6 of 25 dams treated with 20 mg/kg/dayof RDX and occurred between the 11th and the 14th day of gestation. Chronic convulsions occurred in four dams before death.

NEUROTOXICITY
Neurotoxic signs of RDX were observed in 18 of the 25 dams treated with 20 mg/kg/day. These animals showed hyperactivity and other central nervous system related stimulations including convulsions. The appearance of neurotoxic signs in these animals normally occurred on the second day of dosing and then diminished in frequency after the eighth day of dosing. At a dose level of 2.0 mg/kg/day only one female exhibited convulsion during the period of dosing. Convulsions were also observed in one female receiving 350 mg/kg/day of hydroxyurea. There were no neurotoxic signs observed in any females receiving either the vehicle or 0.2 mg RDX/kg/day during the dosing period of day 6-19 of gestation.

LIVER WEIGHT
The absolute liver weight was significantly reduced in dams treated vith 20 mg/kg/day of RDX. There were no meaningful changes in liver weight in dams treated vith low dose levels of RDX or hydroxyurea.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: Embryotoxic effects

Details on embryotoxic / teratogenic effects:
EMBRYOTOXICITY
There were no meaningful changes in number of implants, viable and dead fetuses when the data obtained from treated animals were compared with control animals. High incidence of earlier resorption was noted in surviving animal treated with RDX at 20 mg/kg/day. However, it is difficult to evaluate the significance of this parameter based on only surviving dams.

TERATOGENIC EFFECTS
The external, soft tissue and skeletal anomalies detected in fetuses from rats following oral administration of RDX at 0.2, 2.0, 20.0 mg/kg/day were found to be similar to control rats receiving vehicle only. Hydroxyurea, serving as positive control, produced high incidences of Hydroencephales (soft tissue anomaly), cleft palate, abnormal snout, absence of eye bulges (external anomalies), anomalies of snout, mandible, cranium, vertebrae, sternebrae, axial skeleton and ribs as compared with vehicle or RDX treated rats .

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

2

Dose descriptor:

NOAEL

Effect level:

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

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

See Table 1 below for data on RDX treatment in rats

Table 1 Effect Of Hydroxyurea And RDX Administration During Gestation On Maternal Welfare In Rats

	Hydroxyurea^a	RDX (mg/kg/day)
	350 mg/kg	0	0.2	2.0	20
Females
Treated	24	24	24	24	25
Pregnant (%)	20 (83)	24 (100)	24 (100)	23(96)	24(96)
Deaths (%)	0	0	0	0	6 (25)
Pregnant Survivors	20	24	24	23	17
Day 6 weight (g)	267 ± 2	263 ± 3	265 ± 3	263 ± 3	261 ± 3
Body weight changes (g) on gestation days:
6-7	1 ± 2	4 ± 2	2 ± 1	3 ± 1	-16 ± 2
6-9	9 ± 1	9 ± 2	8 ± 1	10 ± 2	-28 ± 3
9-13	15 ± 2	16 ± 2	19 ± 1	16 ± 2	19 ± 4
Corrected^b	47 ± 3	46 ± 2	49 ± 4	50 ± 4	21 ± 5
Final weight (g)	360 ± 5	369 ± 4	374 ± 6	378 ± 5	324 ± 10
Food consumption (g/day) for gestational days:
6-9	27 ± 2	25 ± 1	25 ± 1	25 ± 1	10 ± 2
9-13	23 ± 2	25 ± 1	26 ± 1	26 ± 1	19 ± 1
13-19	28 ± 1	28 ± 1	27 ± 2	29 ± 1	26 ± 1
Liver weight (g)	135 ± 0.2	13.8 ± 3	13.5 ± 0.4	13.9 ± 0.3	12.3 ± 0.5
(g/kg body weight)	37.7 ± 0.8	37.3  ± 0.6	35.9  ± 0.5	36.7   ± 0.7	37.8  ± 0.6

^a Administered 350 mg/kg/day on gestational day 6, vehicle on days 7 -12

^b Day 20 weight - day 0 weight -weight of reproductive tract and contents

See table 2 below for details on embryo toxicity.

Table 2 Embryo Toxicity

	Hydroxyurea^a	RDX (mg/kg/day)
	350 mg/kg	0	0.2	2.0	20
Implants/Dam	14.8 ± 0.4	14.8 ± 0.3	14.6 ± 0.3	14.7 ± 0.4	13.8 ± 0.5
% Viable fetuses	86.9 ± 4.6	93.2 ± 1.3	97.6 ± 1.2	94.9 ± 1.6	81.4 ± 7.7
% Dead fetuses	0	0.3 + 0.3	0	0	0.4 + 0.4
% early resorptions	6.7 ± 1.6	6.0 ± 1.0	2.5 ± 0.8	4.8 ± 1.4	15.3 ± 7.8
% later resorptions	6.3 ± 4.4	0.5 ± 0.4	0.5 ± 0.3	0.3 ± 0.3	1.6 ± 0.9
Complete resorptions	0	0	0	0	2
Live Litters	20	24	24	23	15
Fetuses/day	12.7 ± 0.7	13.8 ± 0.4	14.2 ± 0.3	14.0 ± 0.4	12.7 ± 0.6
Fetal weight (g)	3.26 ± 0.11	3.63 ± 0.06	3.69 ± 0.08	3.73 ± 0.08	3.36 ± 0.12
% Males	47 ± 4	53 ± 3	47 ± 4	49 ± 3	47 ± 5

Applicant's summary and conclusion

Conclusions:

In this rat teratogenicity study the high dose (20 mg/kg/day) of RDX produced maternal toxicity (neurotoxicity, including convulsions), some maternal deaths and embryotoxicity. The embryotoxicity could be solely the result of the convulsions and consequent hypoxia/transient anoxia. The lower doses (0.2 and 2.0 mg/kg/day) had no adverse effects. Therefore the maternal and developmental NOAEL is 2.0 mg/kg/day. The teratology NOAEL is 20 mg/kg/day.

Executive summary:

HMX and RDX, which is tested for its developmental toxicity in a rat teratogenicity study, are both explosive compounds used in military munitions formulations. These substances have been demonstrated to be very similar in structure, physical/chemical properties and toxicological profile in the Analogue Approach - Read Across High Melting Explosive (HMX) (2013) document (see Section 13).

Due to the fact that HMX and RDX have nearly the same chemical structure, the same mode of interaction with bio-macromolecules, living cells and tissue and metabolic pathway is expected. However, toxicokinetics studies demonstrated that HMX is poorly absorbed while RDX is readily absorbed by the Gastrointestinal tract. This means that the systemic toxic effects of RDX will be more marked than those from HMX and RDX toxicity is a worst case scenario for HMX. Therefore, a read-across from HMX to data obtained with RDX is scientifically justified.