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

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

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. 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 416 (Two-Generation Reproduction 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:

Crj: CD(SD)

Sex:

male/female

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: feed

Details on exposure:

Feed mixes:
Before mixing, RDX was ground in a ball mill to about 200 um particle size, similar to that of the feed. Size was monitored with an American Optical Spencer hemocytometer. This ground RDX was mixed with laboratory rodent chow to provide successive dilution of 10% and 1% RDX in feed. In most cases these were further diluted to provide stock mixtures which were diluted weekly to provide the actual feed mixtures.

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

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. The feed samples were treated with acetonitrile containing RDX, allowed to dry, then reextracted with acetonitrile and assayed by HPLC-UV.

Duration of treatment / exposure:

13 weeks for F0 and F1 male and female rats

Frequency of treatment:

daily

Details on study schedule:

- F1 parental animals not mated until 13 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age (weaning).
- Age at mating of the mated animals in the study: 16 weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
5 mg/kg/day
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
16 mg/kg/day
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
50 mg/kg/day
Basis:
nominal in diet

No. of animals per sex per dose:

F0 generation: 22 animals/sex/dose
F1 generation: 26 animals/sex/dose except for the high dose which contained only one litter of four males and two females.

Control animals:

yes, plain diet

Details on study design:

F0 generation:
Four groups, each consisting of 22 males and 22 females were fed continuously diets which contained quantities of RDX to provide nominal daily doses of 0, 5, 16 or 50 mg/kg.The parenteral generation (F0) was treated for 13 weeks. During this time, weekly body weight, feed consumption, and RDX doses were calculated. After 13 weeks males and females in each group were co-housed in the ratio of one male to one female. During each day of co-housing, females were examined for sperm-positive vaginal smear. If such evidence of mating was observed, the male was removed to a separate cage, the female was weighed, and the day was identified as day 0 of gestation. Mated females were weighed on days 0, 13, and 20 of gestation. Dams were allowed to deliver, and their pups were counted on days 0, 7, 14 and 21 and weighed on days 0 and 4 after birth and again at weaning. Since adverse effects on reproduction were observed in the high dose group, control and high dose females were also mated with non-treated proven male breeders. In addition, F0 males were mated with nontreated females to determine if RDX produced a dominant lethal effect. The results of those matings are in the in vivo genotoxicity robust summary (Cholakis et al., 1980).

F1-generation:
The F1 generation was weaned, and rats were randomly selected from each litter and maintained on the RDX diets. The control, low, and mid-dose groups each consisted of 26 males and 26 females. In contrast, the high dose group contained only one litter of four males and two females. After weaning, each group was fed the appropriate diet for at Ieast 13 weeks. At the end of this treatment, males and females were mated as previously described.

F2 generation:
After the rats were weaned, males and females were randomly selected from litter for necropsy. The tissues listed below were fixed in neutral buffered 10% formalin and processed for histopathological evaluation. All tissues were stained with hematoxylin and eosin.

Examinations

Parental animals: Observations and examinations:

SURVIVAL: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: weekly before mating, mated females were weighed on days 0, 13, and 20 of gestation, pups were weighed on days 0 and 4 after birth and again at weaning

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

No data

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, macroscopy and histopathological evaluation of the weanlings of F2.

Postmortem examinations (parental animals):

No data

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at weaning.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated below were prepared for microscopic examination and weighed, respectively.
Skin, Trachea, Lung, Abdominal Aorta, Heart, spleen,Thymus, Lymph Node (mes.), Kidney, Bladder, Ovary, Uterus, Testicle, Epididymis, Prostate, Seminal Vesicle, Salivary Gland, Esophagus, Stomach, Intestine, Pancreas, Liver, Pituitary, Thyroid, Adrenal, Rib, Diaphragm, Skeletal Muscle, Brain, Spinal cord, Sciatic Nerve, Eye

Statistics:

Quantitative data are reported as the mean standard error and were analyzed for statistical significance by Tukey’s test

Reproductive indices:

Mating = Number mated/cohoused x 100
Fertility = Number fertile (pregnant)/mated x 100

Offspring viability indices:

Viability = Number of pups day 4/day 0 x 100
Lactation = Number of pups day 21/day 4 x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mortality

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

MORTALITY (PARENTAL ANIMALS)
Survival was not affected in the low and mid-dose group.s. In contrast, mortality in the high dose group was 18% during the F0 generation. Marked general toxicity was observed particularly neurotoxicity.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In general the bodyweights of both males and females in the high dose group were consistently reduced in the F0 and F1 generations.In the low and mid dose groups, the bodyweight of the F1 generation was reduced in a few observations however by week 22 and week 27, respectively, there were no significant effect on body weight. Feed consumption was consistently reduced during F0 and F1 generations in both sexes from the high dose group. Intermittent reductions were also observed during F1 generation in males and females from both the low and mid-dose groups; however, these effects were not long lasting.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The concentration of RDX in the diet was varied at weekly intervals in an effort to provide a constant daily dose of the test material as the rats increased in weight. The actual doses of RDX consumed during the study by F0 and F1 males and females is presented in Table 1

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

F0 Mating:
The numbers of males that mated (i.e. l produced sperm-positive vaginal smears in females with which they were co-housed) and were judged to be fertile (i.e., fathered litters with at least one viable pup) were reduced in the high dose group; however, the magnitude of this change did not reach a level of statistical significance, these parameters were normal in the low and mid-dose groups. Adverse effects on reproduction similar to these effects observed in males from the high dose groups were seen in the females with which they were cohoused. In other words, the numbers of females that mated (i.e., had sperm-positive vaginal smears) and were pregnant (i.e., produced litters with at least one viable pup) were reduced in the high dose group; however, this effect, too, was not statistically significant. In addition, the gestational body weight of dams in the high dose group was reduced.

In the second mating of the F0 generation, females from the control and high dose groups were mated with non-treated proven male breeders. Although 80%of the females in both groups mated, the pregnancy rate in both groups was 20%. Since the pregnancy rate was low in both the control and treated groups, it is not possible on the basis of these data to attribute these observations to RDX.

Results of F1 mating:
The numbers of adults, litters, and pups during this mating, the gestational body weight of dams and lactational body weight of their pup were recorded. These observations do not permit meaningful conclusions to be made concerning treatment-related effects in the high dose group because the four males and two females which were available for mating came from the same litters as a result, the number of rats mated was small. Nevertheless, the data do suggest that treatment with the high dose adversely affected pup survival. The gestational body weight of dams in both the mid and high dose groups was significantly reduced.

In the high dose group the fertility, viability, and lactation indexes were reduced.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

16 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical signs; mortality; body weight; food consumption at 50 mg/kg/day

Remarks on result:

other: Generation: P, F1 and F2 (migrated information)

2

Dose descriptor:

NOAEL

Effect level:

16 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: mating index; fertility index at 50 mg/kg/day

Remarks on result:

other: Generation: P and F1 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

effects observed, treatment-related

Details on results (F1)

VIABILITY (OFFSPRING)
The viability of F1 pups, as monitored by the number of litters with at least one viable pup and the number of pups per litter, was reduced in the high dose group. These parameters were not adversely affected in the low and mid-dose groups. In the second mating of the F0 generation, females from the control and high dose groups were mated with non-treated proven male breeders. Although the number of litters was low in both groups, the data do suggest that the high dose of RDX adversely affects pup survival.
No meaningful conclusions could be made concerning treatment-related effects in the F1 high dose group because the four males and two females which were available for mating came from the same litters as a result, the number of rats mated was small. Nevertheless, the F2 data do suggest that treatment with the high dose adversely affected pup survival.

BODY WEIGHT (OFFSPRING)
The body weight of F1 pups in both the mid and high dose groups was reduced 25 days after birth.

ORGAN WEIGHTS (OFFSPRING)
Comparison of the body weights indicated that both the males and females from the mid dose group (16 mg/kg/day) weighed less than the controls. In addition, the absolute weights of male gonads and female kidneys and spleen were significantly reduced. Histopathological examination of the tissues, as described below, did not provide an explanation for these differences.

GROSS PATHOLOGY (OFFSPRING)
Gross observations at the time of necropsy revealed no difference between the experimental groups and the controls.

HISTOPATHOLOGY (OFFSPRING)
Histopathological examination disclosed an increased number of renal tubular epithelial-lined cysts in the cortex of the kidneys of the mid-dose group. Similar cysts were present in both control and low dose groups. The cysts occurred bilaterally mainly in the outer cortex; they were multiple, congenital, benign, and were not associated with renal interstitial inflammatory cell infiltrates, nor with tubular casts or evidence of tubular cell degeneration. They were not caused by intratubular obstruction. They most likely resulted from dysgenesis of nephron formation. The increased number of cysts in the mid-dose animals suggested an RDX-related effect.
Histologic study of the thymus revealed subtle changes in two and a distinct change in a single female of the mid-dose group. While the thymus gland of all control animals was remarkably similar and uniformly normal, the experimental groups showed a slightly greater degree of variability which was considered within the range of normal. The change consisted of a relative diminution of small thymocytes (lymphocytes) in the thymic medulla, exposing the epithelial reticulum and prominent Hassall's corpuscules. A single occurrence of epithelial-lined cyst formation was noted. This was accompanied focally by a severe cytophagocytosis of lymphocytes. The thymic changes observed
were not associated with any alteration of the spleen and lymph nodes. Comparison of control and experimental groups revealed no difference with regard to the latter organs. The thymic changes were not considered significant.

Results: F2 generation

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: an increase in renal cortical cysts and reduced body weights at 16 mg/kg/day

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

See Table 1 below for data from the F0 generation and offspring

.

Table 1: Selected data from F0 parent and F1 offspring during the study.

Nominal RDX Dose (mg/kg/day)
	0	5	16	50
Males
Tested	22	22	22	22
Cohoused	22	22	22	19
Mated	19	21	21	16
Fertile	17	21	20	11
Females
Tested	22	22	22	22
Cohoused	22	22	22	17
Mated	19	21	21	16
Fertile	17	21	20	11
Tested	44	44	44	44
Dead	0	0	0	8 (2M. 6F)
F1 Generation
Liveborn	199	290	255	76
Stillborn (%)	8 (4)	6 (2)	4 (2)	16 (17)
Weaned	173	277	229	6
Tested	52	52	52	6
Dead (%)	0 (0)	1 (2)	2 (4)	0 (0)
Mean weight gain (g) Week 0 to week 13
F0 Male	314	345	302	239
F0 Female	122	123	120	103
Number of Live Litters
Day 0	17	21	19	10
Day 7	16	21	19	4
Day 14	16	21	19	1
Day 21	16	21	19	1
Number of Pups/Litter (Mean ± standard error)
Day 0	11.7 ± 1.0	13.8 ± 0.5	12.8 ± 0.5	7.6 ± 1.4
Day 7	11.3 ± 0.9	13.5 ± 0.5	12.2 ± 0.5	9.0 ± 0.7
Day 14	10.8 ± 0.9	13.3 ± 0.5	11.6 ± 0.6	6.0 ± 0.0
Day 21	10.8 ± 0.9	13.2 ± 0.5	11.5 ± 0.6	6.0 ± 0.0
Dam Body Weight (g)
Day 0	289 ± 6	287 ± 5	290 ± 6	267 ± 7
Day 13	342 ± 7	339 ± 5	339 ± 6	304 ± 9
Day 20	413 ± 11	410 ± 6	403 ± 6	337 ± 11
Pup Body Weight (g)
Day 0	6.6 ± 0.2	6.3 ± 0.1	6.5 ± 0.2	6.2 ± 0.2
Day 4	8.6 ± 0.4	8.5 ± 0.2	8.2 ± 0.2	7.2 ± 0.1
Day 25	53 ± 2	47 ± 2	43 ± 2	41 ± 0

See Table 2 below for selected data from F1 generation and offspring during the study.

Table 2: Selected data from F1 parent and F2 offspring during the study

Nominal RDX Dose (mg/kg/day)
	0	5	16	50
Males
Tested	26	26	26	4
Cohoused	26	26	25	2
Mated	22	24	23	2
Fertile	22	23	20	2
Females
Tested	26	26	26	2
Cohoused	26	25	23	2
Mated	23	24	23	2
Pregnant	22	23	20	2
F2 Generation
Liveborn	282^a	284	248	22
Stillborn (%)	6 (2)	6 (2)	2 (1)	24 (52)
Weaned	223	244	197	0
Necropsied	20	20	20	0
Mean weight gain (g) Week 20 to Week 33
F1 Male	384	381	363	355
F1 Female	210	213	206	192
Number of Live Litters
Day 0	22	23	20	2
Day 7	22	23	19	0
Day 14	21	23	19	0
Day 21	21	23	18	0
Number of Pups/Litter (Mean ± standard error)
Day 0	12.8 ± 0.6	12.3 ± 0.6	12.4 ± 0.8	5.5 ± 1.5
Day 7	11.2 ± 0.7	11.8 ± 0.6	11.8 ± 0.7	-
Day 14	11.6 ± 0.6	11.5 ± 0.6	11.6 ± 0.7	-
Day 21	10.6 ± 0.6	10.6 ± 0.7	11.0 ± 0.7	-
Dam Body Weight (g)
Day 0	255 ± 6	254 ± 5	242 ± 5	236 ± 12
Day 13	308 ± 6	302 ± 5	274 ± 7	275 ± 11
Day 20	380 ± 6	368 ± 6	336 ± 9	311 ± 3
Pup Body Weight (g)
Day 0	6.2 ± 0.1	6.3 ± 0.1	6.1 ± 0.1	7.4 ± 2.0
Day 4	7.9 ± 0.3	8.3 ± 0.3	7.4 ± 0.2
Day 21	30 ± 1	31 ± 1	26 ± 1

^a Number illegible in original report. Data estimated from number of live litters and average number of pups per litter lower down in the table.

Applicant's summary and conclusion

Conclusions:

In this rat two-generation reproduction study, doses up to 50 mg/kg/day had marked general toxicity, but no specific reproductive effects other
than those which could be ascribed to poor nutrition from the general toxicity. Mid dose group (16 mg/kg/day) produced no apparent toxicity but the histopathological evaluation of the F2 generation showed a RDX-related finding of statistical significance: an increase in renal cortical cysts (there were no F2 weaned rats at the high dose due to low mating numbers and survival). The reproduction and general NOAEL is 5mg/kg/day

Executive summary:

HMX and RDX, which is tested for its toxicity to reproduction in a rat 2 generations 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.