Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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 .
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals 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
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
Remarks:
Doses / Concentrations:
5 mg/kg/day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
16 mg/kg/day
Basis:
nominal in diet
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.

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
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: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
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.
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)
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)
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
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.

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
Reproductive effects observed:
not specified

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

 282a

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.

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.
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
5 mg/kg bw/day
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

This study 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 .


Short description of key information:
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 NOAEL is 5mg/kg/day

Justification for selection of Effect on fertility via oral route:
Only 2-generation reproduction study available. This study was performed before GLP and OECD guidelines. However the method used is equivalent to OECD guideline 416.

Effects on developmental toxicity

Link to relevant study records
Reference
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 .
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Fischer 344
Details on test animals 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
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
Remarks:
Doses / Concentrations:
0.2 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
2 mg/kg/day
Basis:
actual ingested
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.
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.
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.


Dose descriptor:
NOAEL
Effect level:
2 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
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 .
Dose descriptor:
NOAEL
Effect level:
2 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

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

 

Hydroxyureaa

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

Correctedb

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

 

Hydroxyureaa

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

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.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
2 mg/kg bw/day
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The potential developmental toxicity of RDX was examined in three studies, two in rats and one in rabbits (Cholakis et al., 1980; Angerhofer, 1986). In the first study, pregnant Fisher 344 rats were dosed by gavage with RDX in 1% methylcellulose and 1% polysorbate 80 aqueous solutions at 0.2, 2 and 20 mg/kg/day from Day 6 through to Day 19 of gestation (Cholakis et al., 1980). In the second study, Sprague-Dawley rats were dosed by gavage with RDX in 10% aqueous gum acacia at 2, 6 and 20 mg/kg/day from Day 6 to Day 15 of gestation (Angerhofer, 1986).

25% and 31% maternal deaths, neurotoxic signs, reduced bodyweight gain and food consumption were observed in both rat studies at the highest dose tested. Cholakis (1980) reported that RDX had no effects on the number of implants, viable and dead foetuses. RDX was embryotoxic at the maternally toxic dose of 20 mg/kg/day - higher incidence of early resorption was noted in surviving animals treated with RDX at the highest dose. Angerhofer (1986) reported a decrease in foetal weight and length at all RDX doses when data were analysed on an individual basis. However when analysed on a litter basis, decreases in fetal weights and lengths were only significant at the 20 mg/kg/day dose level. In neither study were increases in the occurrence of foetal malformations observed at doses up to 20 mg/kg/day (Cholakis et al., 1980; Angerhofer, 1986).

In the rabbit teratology study, New Zealand rabbits were dosed by gavage with RDX in 1% methylcellulose and 1% polysorbate 80 aqueous solutions at 0.2, 2 and 20 mg/kg/day from Day 7 through to Day 29 of gestation (Cholakis et al., 1980). Dams receiving 20 mg/kg/day of RDX gained less weight during gestation than controls. However, the treatment with RDX did not affect the percent of viable or dead foetuses and the incidence of early and late resorption. The number of foetuses per dam and fetal weight were normal in all groups treated with RDX. This study revealed no foetotoxicity at doses up to 20 mg/kg/day; the rabbits were less sensitive to the general toxicity of RDX than the rats.


Justification for selection of Effect on developmental toxicity: via oral route:
This study was performed before GLP and OECD guidelines. However the method used is equivalent to OECD guideline 416.

Justification for classification or non-classification

No reproductive or developmental toxicity studies are available on HMX. However, in the 90 day repeat dose studies, no gross or histopathological lesions were observed in the uterus or ovaries of female rats and mice exposed to 1511.9 and 93.1 mg HMX/kg/day, respectively, or testes of male rats and mice exposed to 4101.3 and 75.1 mg HMX/kg/day (Everett et al, 1985; Everett and Maddock, 1985). Data is available on analogue RDX via the oral route from a two-generation reproductive study in rats, a dominant lethality assay in rats, two developmental toxicity studies in rats and one in rabbits (Cholakis et al., 1980; Angerhofer, 1986). The data suggest that at non-toxic doses, RDX is not a reproductive or developmental toxicant. Therefore HMX is not classified in accordance with Regulation (EC) No 1272/2008.