Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Name of test substance: Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen
CAS No. 13897-55-7 and 13897-56-8
Test substance No: 10/0117-5
Batch identification: B4360
Purity: 99.762%
Homogeneity: given (study code 15L00360)
Stability: stable until 21 Jul 2017

Test animals

Species:
rat
Strain:
Wistar

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
GD6-19 (14 applications)
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
DOSE RATIONAL
In a previous OECD 422 - Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats with Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen (project no. 85R0117/10S114, see also IUCLID section 7.5.1 and 7.8.1), doses of 25, 100 and 250/200 mg/kg bw were administered by gavage to 10 animals per group and sex. At 250 mg/kg bw/d, clinical findings (semiclosed eyelids, salivation, respiratory sounds), reduced food consumption (females: -19 % compared to control in study week 1) and a decrease in body weight change (females: -81 % compared to control in study week 1) were observed. The high-dose group was lowered on study day 7 of treatment due to pronounced toxicity. At 200 mg/kg bw/d, one female was found dead on study day 30 (GD 4) and another one was sacrificed moribund on study day 20 (GD 4). Clinical findings (salivation, piloerection, semiclosed eyelids, slight labored respiration, respiratory sounds, reduced general condition, hypothermia and poor general state) were still observed during the treatment period. Food consumption and body weights were decreased. Reproductive performance, clinical pathology and pathology of the animals showed no adverse changes. At the next lower dose of 100 mg/kg bw/d, one female animal was found dead on study day 36 (GD 21). No further parameter showed adverse changes. At this time, it was not possible to assess the cause of a single premature death at a dose level of 100 mg/kg bw/d. Applying a precautionary principle a link between this observation and test substance administration was assumed. Due to signs of systemic toxicity, the female no observed effect level (NOEL) for general toxicity was the lowest tested dose of 25 mg/kg bw/d.

Based on the available data and at the request of the sponsor, the following dose levels were chosen for the present prenatal developmental toxicity study in Wistar rats:
5 mg/kg body weight/day as low-dose level
25 mg/kg body weight/day as mid-dose level
100 mg/kg body weight/day as high-dose level

The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.

Examinations

Maternal examinations:
Mortality
A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20).

Clinical symptoms
A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. During the administration period (GD 6-19) all animals were checked daily for any abnormal clinically signs before administration as well as within 2 hours and within 5 hours after administration.

Food consumption
The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13- 15, 15-17, 17-19 and 19-20.

Body weight data
All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results.

Corrected (net) body weight gain
Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).

Clinical Pathology
In the morning blood was taken from the retro-bulbar venous plexus from non-fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units.

Pathology
Necropsy
On GD 20 all surviving dams were sacrificed by decapitation under isoflurane anesthesia in a randomized sequence. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

Organ weights
The following weights were determined in all animals sacrificed on schedule:
1. Adrenal glands
2. Kidneys
3. Liver
4. Spleen

The carcass weights (GROSSE-System) were transferred to the ACOPAT-System to calculate the relative organ weights.

Organ / Tissue fixation
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Kidneys
4. Liver
5. Spleen

No further examinations or procedures were performed in the study.
Ovaries and uterine content:
Cesarean section
On GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in randomized order. After the dams had been sacrificed, they were necropsied and assessed for gross pathology as described in chapter 3.9.2. “Pathology”. The uteri and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
• Live fetuses
• Dead implantations:
a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy)
b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

After the weight of the uterus had been determined, all subsequent evaluations of the dams and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.
Fetal examinations:
EXAMINATIONS OF THE FETUSES
All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.

Examinations of the fetuses after dissection from the uterus
At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus). After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half was placed in Harrison’s fluid for fixation.

Soft tissue examination of the fetuses
The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR. After this examination these fetuses were discarded.

Skeletal examination of the fetuses
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were retained individually.

Evaluation criteria for assessing the fetuses
In the present study the glossary of WISE et al. (1997) and its updated version of MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD et al. (1999) and SOLECKI et al. (2001, 2003):
Malformation
A permanent structural change that is likely to adversely affect the survival or health.
Variation
A change that also occurs in the fetuses of control animals and/or is unlikely to adversely affect the survival or health. This includes delays in growth or morphogenesis that have otherwise followed a normal pattern of development. The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations. All fetal findings were listed in tables according to these classifications.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 5, 25 or 100 mg/kg bw/d during the entire study period.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any females of all testgroups (0, 5, 25 or 100 mg/kg bw/d).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights and average body weight gain of the low-, mid- and high-dose dams (5, 25 or 100 mg/kg bw/d) were in general comparable to the concurrent control group throughout the entire study period.

The corrected body weight gain of test groups 1, 2 and 3 (5, 25 and 100 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group. Moreover, mean carcass weights remained also unaffected by the treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The mean food consumption of the dams in test groups 1, 2 and 3 (5, 25 and 100 mg/kg bw/d) was comparable to the concurrent control throughout the entire study period. The statistically significantly increased food consumption value in test group 1 on GD 6-8 is assessed as incidental.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At gestation day 20, in dams of test group 3 (100 mg/kg bw/d) relative monocyte counts were significantly higher compared to controls. The absolute monocyte counts were also marginally above the historical control range although not significantly increased (relative monocytes 2.0-2.9 %; absolute monocytes 0.08-0.16 giga/L). Neither any other differential blood cell count nor total white blood cell counts were changed. Therefore, this isolated change of the monocytes was regarded as maybe treatment-related but not adverse (ECETOC Technical Report No. 85, 2002).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At gestation day 20 in dams of test group 3 (100 mg/kg bw/d) creatinine values were lower compared to controls. However, the mean was within the historical control range (creatinine 22.8-31.5 μmol/L). Therefore, this alteration was regarded as incidental and not treatmentrelated.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
All mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Maternal developmental toxicity

Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Only pregnant dams were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant dams with scheduled sacrifice (GD 20) were used for the calculation of mean gravid uterine weights, corrected (net) body weight gain and summary of reproduction data. The following females were excluded from the above-mentioned calculations:
Test group 0 (0 mg/kg bw/d):
• female No. 4 – not pregnant
Test group 2 (25 mg/kg bw/d):
• female No. 71 – not pregnant

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the concurrent control group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (5, 25 and 100 mg/kg bw/d) was comparable to the control fetuses.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
For one female mid-dose fetus (No. 70-07; 25 mg/kg bw/d) a gastroschisis was recorded during external observation. Since there was no relation to dosing, this finding was assessed as incidental. The overall incidences of external malformations were comparable to those found in the historical control data.

One external variation in one single fetus (No. 89-06) was recorded in test group 3 (100 mg/kg bw/d), i.e. limb hyperextension. The incidence of this single finding was not statistically significantly different from control and it can be found in the historical control data at comparable or higher incidences (HCD of affected fetuses per litter: mean 0.0 %, [0.0-0.7 %]). Thus, it is not considered as treatment-related and adverse.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Some fetuses of test groups 0, 1 and 3 (0, 5 or 100 mg/kg bw/d) had skeletal malformations. The findings in the high-dose group occurred in one single fetus each and were also covered by the historical control data with comparable incidences. The combination of “shortened scapula” and “shortened humerus” was also seen in one control fetus. Therefore, the high-dose findings were not assessed as treatment-related and adverse. All other malformations were not related to dosing and, therefore, assessed as not treatment related. The total incidence of skeletal malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data.

For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dosing. The overall incidences of skeletal variations were comparable between treated groups and concurrent control as well as with the historical control.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One single high-dose fetus had soft tissue malformations. The findings can be found also in the historical control data with comparable incidences (HCD of affected fetuses per litter: 0.0 %, [0.0-0.8%]). Thus, it is not considered as treatment-related and adverse.

Three soft tissue variations were detected in all test groups including the control (0, 5, 25 or 100 mg/kg bw/d), i.e. short innominate, dilated renal pelvis and dilated ureter. The incidences of these variations were neither statistically significantly different from control nor dose-dependent and, therefore, not considered biologically relevant. All of them can be found in the historical control data at comparable incidences.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the vertebral column, the ribs and the sternum and did not show any relation to dosing. The overall incidences of skeletal unclassified cartilage observations in the substance-treated groups did not differ significantly from the concurrent control group.
Details on embryotoxic / teratogenic effects:
External (Tab. ID-003), soft tissue (Tab. ID-006) and skeletal (Tabs. ID-012 - ID-013) malformations
were noted in all test groups (0, 5, 25 and 100 mg/kg bw/d).
Three fetuses were multiple malformed: male control fetus No. 24-06, as well as male highdose
fetus No. 87-06 (100 mg/kg bw/d), had skeletal malformations concerning the upper limbs
(i.e. shortened scapula, shortened humerus). For male high-dose fetus No. 84-06 (100 mg/kg
bw/d) a hydronephrosis combined with a hydroureter was recorded, while male high-dose fetus
90-06 (100 mg/kg bw/d) had severely malformed skull bones (such as split basisphenoid, misshapen
presphenoidal bone, partly absent palatine bones). No ontogenetic pattern is recognizable
for the individual malformations nor was there any cluster of any of these individual
malformations seen in the other offspring of the high-dose group. All of them are present in the
historical control data of the rat strain. Thus, a relationship of these three cases of malformed
fetuses to the treatment is not assumed.
Othermalformations, i.e. gastroschisis, split scapula andmalpositioned and bipartite sternebra,
were not related to dose and all of them can be found in the historical control data. An association
of these findings to the treatment is also not assumed.
One external variation (Tab. ID-004), some soft tissue variations (Tabs. ID-008 - ID-009) and
a range of skeletal variations (Tabs. ID-014 - ID-025) occurred in all test groups including the
controls. None of the total incidences showed a relation to dosing (see Tab. 4.3.5.2.). The
majority of individual variations were equally distributed about the different test groups, if normal
biological variation is taken into account, and can be found in the historical control data at
a comparable frequency.
No unclassified external and no unclassified soft tissue observations were recorded for any of
the fetuses in this study. A spontaneous origin is assumed for the unclassified skeletal cartilage
observations (Tabs. ID-026 - ID-028) which were observed in several fetuses of all test groups
(0, 5, 25 and 100 mg/kg bw/d). The distribution and type of these findings do not suggest any
relation to treatment.
Finally, fetal examinations revealed that there is no effect of the compound on the respective
morphological structures up to a dose of 100 mg/kg bw/d.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEC
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at a dose of 100 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicology is 100 mg/kg bw/d.
Executive summary:

In a prenatal developmental toxicity study the test substance Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity. Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle. Generally, clinical observations including food consumption and body weight gain revealed no toxicologically relevant difference between the animals receiving 5, 25 or 100 mg/kg bw/d Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen and controls. Regarding clinical pathology, no treatment-related, adverse effects were observed up to a dose of the test substance of 100 mg/kg bw/d. Regarding pathology, all findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. No differences of toxicological relevance between control and the treated groups were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose. Fetal external, soft tissue and skeletal examinations revealed that there is no effect of the compound on the respective morphological structures up to a dose of 100 mg/kg bw/d.