Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD TG 422 under GLP conditions (BASF SE, 2011), oral, rats: NOAEL = 200 mg/kg bw/d

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-06-21 - 2011-08-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (GLP)
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Wistar Rat; Crl:WI(Han) from Charles River Laboratories, Research Models and ServiceGmbH, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks at arrival, 11-12 weeks after acclimation
- Weight at study initiation: means of 320-322 for males and 203-204 g for females
- Housing: individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²). During overnight matings, male and female mating partners were housed together in Makrolon type M III cages. For motor activity (MA) measurements the animals were housed individually in polycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material (the present supplier is documented in the raw data).
- Diet: ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: drinking water (from water bottles); ad libitum
- Acclimation period: ca 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
highly deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, the vehicle was filled up to the desired volume, subsequently released with a magnetic stirrer. The test substance preparations were produced at least once a week.

VEHICLE
- Concentration in vehicle: 0, 0.25, 1.0 and 2.5/2.0 mg/100 mL, respectively in the 0, 25, 100 and 250/200 mg/kg bw dose groups
- Amount of vehicle (if gavage): the administration volume was 10 mL/kg body weight.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: the animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning, for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): pregnant animals and their litters were housed together until PND 4 (end of lactation).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The test substance was completely miscible with highly deionized water and, thus, the test substance preparation was considered to be homogenous.
- The stability of the test substance in highly deionized water for a period of 7 days at room temperature was proven before the start of the study.
- The analyses (spectropic [H-NMR], chromatographic [HPLC / GC] and titration [GC/MS] methods) were carried out in compliance with the Principles of Good Laboratory Practice as a separate study at the test facility.
Duration of treatment / exposure:
38 days for males, 52 days for females; the duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females.
Frequency of treatment:
Daily
Details on study schedule:
- Age at mating of the mated animals in the study: 13-14 weeks
Remarks:
Doses / Concentrations:
25, 100 and 250/200 mg/kg bw per day (the high dose was reduced from 250 to 200 mg/kg bw from day 7 onwards)
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
None
Parental animals: Observations and examinations:
See IUCLID Chap. 7.5.1 Repeated dose toxicity

OTHER:
- The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis.
- On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.
- The day of littering was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 0) and on PND 4. Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly, and the determined body weight data were solely used for the calculations of the dose volume.
Sperm parameters (parental animals):
Parameters examined in all male parental generations: testis, cauda epididymis, prostate, epididymides, seminal vesicles weights
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain (PND1-PND4), physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS: yes
Postmortem examinations (parental animals):
See IUCLID Chap. 7.5.1 Repeated dose toxicity
After sacrifice of the female animals the uterus and ovaries were removed and the implantation sites were counted. To determine the number of implantation sites in apparently non-pregnant animals, the uteri from those females were stained in 10% ammonium sulfide solution for about 5 minutes according to the method of SALEWSKI. Then, the respective uteri were rinsed carefully with fresh tap water. The implantation sites were recorded for the calculation of the post-implantation loss.
Postmortem examinations (offspring):
SACRIFICE
All surviving pups (sacrificed on PND 4 under isoflurane anesthesia with CO2)

GROSS NECROPSY
All stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically. All pups were discarded after their evaluation.
Statistics:
- Means, medians and standard deviations of each test group were calculated for several parameters.
- Number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, mean litter weight and number of pups delivered per litter were analyzed by simultaneous comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means.
- Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index and number of litters with affected pups at necropsy were analyzed by pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions
- Proportions of affected pups per litter with necropsy observations were analyzed by pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
Reproductive indices:
Male fertility index, female mating index, female fertility index, female gestation index, live birth index and post implantation loss.
Offspring viability indices:
Viability index, sex ratio
CLINICAL SIGNS AND MORTALITY
1) Mortality
- Female animals: 1 female animal of test group 3 (250 / 200 mg/kg bw/d) was found dead on study day 30 (gestation day 4; GD 4). 1 further female animal of test group 3 was sacrificed moribund on study day 20 (GD 4). 1 female animal of test group 2 (100 mg/kg bw/d) was also found dead on study day 36 (GD 21).
2) Clinical signs
- During gestation: Semi-closed eyelid in both eyes after treatment was observed in all animals of test group 3 from GD 0 onwards. In addition, 1 animal of test group 3 showed hypothermia on GDs 1 and 2, poor general state (moderate) from GD 1 to 4, labored respiration from GD 0 to 4 and piloerection from GD 1 to 4. Furthermore, 1 additional animal of test group 3 showed poor general state, labored respiration, respiratory sounds and piloerection during the first week of gestation. All findings were assessed as being related to treatment.
2 sperm-positive female animal of the test group 3 and one of test group 2 did not deliver F1 pups.
- During lactation: semi-closed eyelid in both eyes after treatment was observed in all animals of test group 3 from PND 0 onwards. The finding was assessed as being related to treatment.

BODY WEIGHT AND WEIGHT GAIN
- During gestation: mean body weight was significantly lower by -13% on GD 20 in the test group 3. The same was true for body weight change values between GD 7 and 14 (-21%), GD 14 and 20 (-44%) as well as for the entire gestation period (-31%).
- During lactation: mean body weight was significantly lower on PND 0 as well as on PND 4, with a maximum of -10% on PND 0 in the test group 3. However, body weight change was not impaired. All findings were assessed as being related to treatment.
No changes were observed in female animals of test groups 1 and 2 (25 and 100 mg/kg bw/d) over the entire administration period.

FOOD CONSUMPTION
- During gestation: after changing the dose levels to 200 mg/kg bw/d food consumption was significantly decreased in female animals during the gestation period, i.e. -14% on GD 20 and lactation period, i.e. -12% on PND 4. These findings were assessed as being related to treatment.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
1) Males reproduction data
- Male mating index: with the exception of 2 male animals of test group 0 (0 mg/kg bw/d; control group) and 2 of test group 1, mating was confirmed for all F0 parental males, which were placed with females to generate F1 pups. Thus, the male mating index was 100% in test groups 2 and 3, and 80% in test groups 0 and 1.
- Male fertility index: fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. Two control males, 2 male animals of test group 1, 1 male animal of test group 2 and 2 male animals of test group 3 did not generate F1 pups. Thus, the male fertility index ranged between 80% and 90% (80% in test groups 0, 1 and 3, and 90% in test group 2). These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data
2) Females reproduction data
- female mating index: The female mating index calculated after the mating period for F1 litter was 80% for test groups 0 and 1 and 100% for test groups 2 and 3. The mean duration until sperm was detected (GD 0) was 3.8, 2.5, 2.2 and 3.7 days in test groups 0, 1, 2 and 3, respectively.
- Female fertility index: all sperm positive rats delivered pups or had implantation sites with the exception of 2 female animals of the control group, 2 of test group 1, 1 of test group 2 and 2 of test group 3. These animals were either sperm-negative or did not become pregnant. Thus, the female fertility index varied between 80% and 100% (100% in test groups 0 and 1, and 90% in test group 2 and 3).
- Gestation index: the gestation index reached only 75% in test group 3, 89% in test group 2, 88% in test group 1 and 100% in control group.
- Live birth index: the total number of delivered pups was significantly decreased. However, the relative amount of liveborn pups was not affected by the test substance, as indicated by live birth indices of 100% (test groups 0, 1 and 2) and 98% (test group 3). A single stillborn pup was only seen in test group 3 but assessed as being incidental.

GROSS PATHOLOGY
- The seminal vesicles of 4/10 male animals of test group 2 and 4/10 male animals of test group 3 showed slight enlargement.
- The mating pairs without litters did not show relevant gross lesions except for those animals with slightly enlarged seminal vesicles (see above). Since the number of mating pairs with no litters was equally distributed between control animals and test groups, the enlarged seminal vesicles were considered not to be the reason for reduced fertility in these animals. This assumption was supported by the fact, that all other mating pairs of test group 2 and 3 had a normal outcome of offspring although the male animals showed slightly enlarged seminal vesicles.
- 1 female animal of test group 3 which died ahead of schedule, showed a moderate dilation of duodenum, jejunum, ileum, cecum and colon and a severe dilation of the stomach. A further female animal of test group 3 was sacrificed in a moribund state and showed a severe dilation of jejunum and cecum. Dilation of the gastrointestinal tract in both animals was considered to be a postmortal and/or agonal change due to bacterial gas formation and was not considered to be treatment-related.
All other gross findings noted at necropsy were regarded as incidental and spontaneous in origin and were not related to the treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
- 2 female animal of test group 3 showed a diffuse thymic atrophy. Since the animals were sacrificed in a moribund state or were found dead, the thymic atrophy was considered to be the result of a general poor condition.
- Two female animals of test group 3 showed a minimal (grade 1) follicular hypertrophy/hyperplasia of thyroid glands. Since hypertrophy/hyperplasia was only minimal and since only 2 female animals were affected this finding was considered to be incidental in origin and not treatment-related. This conclusion was supported by the fact, that there was no significant increase in thyroid gland weights, in contrast, the relative weights of thyroid glands in females of test group 3 were slightly decreased. Neither macroscopic examination nor histopathologic examination of the animals that died ahead of schedule or were sacrificed in a moribund state revealed any pathological finding that would explain the death and the poor general condition, respectively.
All other histopathologic findings noted were either single observations, or were biologically equally distributed between control animals and treated rats. All of them were considered to be incidental and spontaneous in origin.

HISTORICAL CONTROL DATA (if applicable)
Historical control data were available and were used for the evaluation of the biological significance of the observed changes.
Dose descriptor:
NOEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on mortality (pregnant female) in the 100 mg/kg dose group at the end of the gestation period
Dose descriptor:
NOAEL
Remarks:
male and non-pregnant female
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
mortality
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Highest dose evaluated
VIABILITY (OFFSPRING)
The viability index as indicator for pup mortality between PND 0 and 4 was 99% for the control, 100% for test groups 1 and 2 and 98% for test group 3 (1 pup was cannibalized). These findings were assessed to be incidental and not related to treatment.
The mean number of delivered pups per dam was significantly decreased in test group 3 (mean value of 9.0). The finding was assessed as being incidental as the calculation based on only 6 litters and negatively influenced by one dam, which delivered only 4 pups (for comparison, 1 female animal of group 1 also had only 5 pups but the mean value of test group 1 was 12.7). In addition, the lower bound for this parameter in the historical control data is very close to 9.0, i.e. 9.3). For all test groups the rate of liveborn and stillborn pups reflect the normal range of biological variation inherent in the strain used in this study.

CLINICAL SIGNS (OFFSPRING)
The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.
The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences between test groups.

BODY WEIGHT (OFFSPRING)
Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the concurrent control values.

GROSS PATHOLOGY (OFFSPRING)
Dextrocardia was observed in 1 pup of test group 1. A discolored liver (dark red) was observed in each 1 pup of test group 2 and 3. These findings were assessed as being spontaneous in nature and without biological relevance.
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Highest dose evaluated
Reproductive effects observed:
not specified

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration by gavage of the test substance to male and female Wistar rats revealed signs of systemic toxicity at a dose level of 250/200 mg/kg bw/d.

-Female animals (dams): 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.

- Male animals: the coagulating glands and seminal vesicles of male animals of test groups 2 and 3 showed minimal to mild dilation with flattening of lining epithelial cells. Findings in coagulating glands correlated well with increased absolute and relative weights, as they were weighted together with seminal vesicles, whereas histopathological findings of seminal vesicles correlated to macroscopically enlarged glands and increased absolute and relative weights. Minimal to mild dilation with flattening of lining epithelial cells in dorsolateral lobes of prostate glands was additionally seen in males of test groups 1 to 3, as well as increased size of ventral lobes of prostate glands of animals of test groups 1 to 3. Histopathological findings of the prostate glands correlated also with increased absolute and relative weights, although there was no clear dose-response relationship especially in absolute weight parameters (probably because of the reduced body weight of test group 3 males).

As the study results did not reveal any impact of the substance on reproduction, it has been concluded, that increased filling of accessory sex glands did not influence reproductive functionality and was therefore considered to be a treatment-related, but non-adverse finding.

Epididymides (test group 3), and epididymal tails (test group 2 and 3) showed slight, but significantly increased relative weights. Although there were no corresponding histopathologic findings in this organ, a treatment-related effect was assumed, since the mean relative weights of epididymal tails were beyond historical control data.

Because the weight change of epididymides and epididymal tails did not have any impact on reproduction, it has been concluded, that increased relative weights of epididymides and epididymal tails did not influence reproductive functionality and was therefore considered to be non-adverse.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
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

The potential of the test substance to induce toxicity to reproduction after administration to test animals was evaluated in a study conducted according to the OECD TG 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) under GLP conditions (BASF SE, 2011; also see IUCLID section 7.5.1 Repeated dose toxicity). After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear.

During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, day of parturition (postnatal day [PND] 0) and on PND 4. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed with CO2 and examined macroscopically for external and visceral findings.

Following relevant test substance-related results were obtained only for parental animals:

1) For the test group 3: 1 female animal was found dead on study day 30 (GD 4) and another one was sacrificed moribund on study day 20 (GD 4). Semi-closed eyelids after treatment were also observed in 1 or both eyes of all animals during gestation and lactation period; the finding was also observed in 2 of 5 females at home cage observation during functional observational battery. Labored respiration, respiration labored slight, respiratory sounds, reduced general condition, hypothermia and poor general state on several days were observed in 2 of 10 female animals during the gestation period, as well as piloerection in 2 of 10 females from GD 1 to 4. Food consumption was significantly decreased in female animals by -14% on GD 20; and by -12% on PND 4 (accompanied by decreased body weights parameters during the entire study period).

1) For the test group 2: 1 female animal was found dead on study day 36.

Dilation (with corresponding histopathologic finding) of coagulating glands (test groups 2 and 3), seminal vesicles (test groups 2 and 3), lateral lobes of prostate glands (test groups 1 to 3) and the increased size of ventral lobes of the prostate glands (test groups 1 to 3), as well as slight, but significantly increased relative weights (without corresponding histopathologic finding) of Epididymides (test group 3) and epididymal tails (test group 2 and 3) were observed in male animals and considered to be treatment-related, although, there was no hint to a specific toxicological mechanism. However, because these changes did not have any impact on reproduction, they were therefore considered to be non-adverse.

No further test substance-related adverse effects were observed in the test groups 1, 2 or 3, regarding clinical pathology, pathology, and reproduction performance or in pups at clinical examinations/ gross findings.

Therefore, under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the LOAEL of the test substance was 100 mg/kg bw/d (based on the mortality of one dam at the end of gestation) in rats. The NOAEL for reproduction performance, fertility and developmental toxicity, was set to 200 mg/kg bw/d in male and female Wistar rats.


Effects on developmental toxicity

Description of key information
OECD TG 414 under GLP conditions (BASF SE, 2017), oral, rats: NOAEL 100 mg/kg bw/d
Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
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
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.
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.
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.
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
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
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.
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
Abnormalities:
not specified
Key result
Developmental effects observed:
no
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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
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

Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered as an aqueous solution to groups of 25 time-mated female Wistar rats by gavage at doses of 5, 25 and 100 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (ultrapure water) in parallel.

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.

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.

Justification for classification or non-classification

Classification,Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for toxicity to reproduction under Regulation (EC) No. 1272/2008, as amended for the third time in Directive (EC 618/2012).