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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because a pre-natal developmental toxicity study is available
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 October 2022 to 9 March 2023
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 18-20 weeks weeks
- Weight at study initiation: 3071 – 4622 g
- Fasting period before study: no
- Housing: individual
- Diet (e.g. ad libitum): KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding (Granovit AG, Kaiseraugst, Switzerland); restricted access: approximately 25 grams pelleted diet and on subsequent days 170-190 grams; in addition, pressed hay and fresh vegetables were provided

- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5-6

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 19°C
- Humidity (%): 53 to 84%.
- Air changes (per hr): at least 10 per hour
- Photoperiod (hrs dark / hrs light): 12-hours light and 12-hours dark

IN-LIFE DATES: From: 11 October 2022 To: 11 November 2022
Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous carboxymethyl cellulose (CMC) + 0.1% Tween80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: at least weekly

VEHICLE
- Justification for use and choice of vehicle (if other than water): addition of CMC and Tween80 for a stable suspension
- Concentration in vehicle: 0, 10, 20, 40 mg/mL
- Amount of vehicle (if gavage): 5 ml/kg
Analytical verification of doses or concentrations:
yes
Remarks:
Analysis was performed with an ultra performance liquid chromatographic method with spectrophotometric detection (UPLC-UV).
Details on analytical verification of doses or concentrations:
Accuracy and homogeneity were determined for formulations prepared for use during treatment in Week 1.
The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e., mean sample concentration results were within or equal to 85-115% of target concentration).
A small response at the retention time of the test material was observed in the chromatograms of the Group 1 (vehicle control) formulation. It was considered not to derive from the formulation since a similar response was obtained in the analytical blanks. The maximum contribution to the Group 2 samples was 0.18%, which was considered negligibly low for the purposes of this study.
Details on mating procedure:
Time-mated females; untreated females were mated at the supplier and were at Day 1 or 2 post-coitum on arrival at the test facility (Day 0 post-coitum is the day of successful mating).
Duration of treatment / exposure:
Day 7 to Day 28 post coitum
Frequency of treatment:
daily
Duration of test:
scheduled euthanasia at day 29 post-coitum
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were based on the results of the dose range finding study (Test Facility Study No. 20342602).
- Rationale for animal assignment (if not random): random; prior to dosing female Nos. 23 and 67 originally allocated to the 50 and 200 mg/kg/day group, respectively, were interchanged between the groups, due to poor acclimatization of Female No. 67 (i.e., (almost) absent food consumption from first recording on Day 3 post-coitum onwards)
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily (0 to 1 hours post-dose)

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 3, 7, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.

FOOD CONSUMPTION: Yes (daily from Day 3 post-coitum onwards)

WATER CONSUMPTION: Yes
- Time schedule for examinations: regular (visual inspection of the water bottles without reporting)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 29 post-coitum
- Organs examined: external, thoracic and abdominal examination, with special attention to the reproductive organs; each ovary and uterine horn of all animals was dissected and examined; histology and microscopic evaluation of the gastrointestinal tracts including stomach, duodenum, jejunum, ileum, caecum, olon, rectum, appendix and sacculus rotondus for the animals from Group 1 and 4.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes

Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number and distribution of live and dead fetuses, placental morphology
Blood sampling:
No
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [all per litter ]
- Skeletal examinations: Yes: [all per litter ]
- Head examinations: Yes: [50% per litter underwent soft-tissue examination and 50%were examined by mid-coronal slice]
Statistics:
see attached document "20342615 Statistical Analysis"
Indices:
Pregnancy Rate (%)
Live Male Fetus/Litter (%)
Live Female Fetus/Litter (%)
Pre-Implantation Loss (%)
Post-Implantation Loss (%)
Litter % of Fetuses with Abnormalities
Historical control data:
see attached documents
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No test material-related clinical signs were noted during the treatment period up to 100 mg/kg/day.

At 200 mg/kg/day, one female was observed with slight tremors on Day 26 post-coitum and another female with increased respiratory rate on Day 27 post-coitum. Based on the similarities with findings in the previous dose range finder study (see discussion for further details) this was considered test material-related.
Erected fur was noted for 1/20 and 2/21 females at 100 and 200 mg/kg/day, respectively, on one to three days between Days 17-20 post-coitum. At the isolated/very low incidence and due to its transient nature, this finding was considered not related to treatment with the test material.
Slight to severe salivation was seen after dosing for 11/21 animals at 200 mg/kg/day, mainly in the third week of dosing. Taking into account the nature and in general minor severity of the effect and its time of occurrence (i.e., after dosing), this sign was considered to be a physiological response rather than a sign of systemic toxicity.
Other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test material.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
In total, three animals (two in the 100 mg/kg/day group and one in the 200 mg/kg/day group) did not survive until scheduled necropsy. Only the unscheduled death in the 200 mg/kg/day group was considered test material related. The remaining two preterm deaths were considered related to the gavage procedure.

Test material-related mortality:
At 200 mg/kg/day, Female No. 85 was sacrificed in extremis for animal welfare reasons on Day 18 post-coitum, as it was noted with (nearly) absent food consumption for eight consecutive days (Days 10-18 post-coitum). The animal was also noted with a 2% body weight loss over Days 7-15 post-coitum, followed by a 3% body weight gain over Days 15-18 post-coitum. No clinical signs were noted on previous days, and necropsy did not reveal any abnormalities. This animal was pregnant with ten embryos in development corresponding with the duration of the pregnancy.

Mortality unrelated to treatment with the test material
There were two mortalities (both at 100 mg/kg/day) that were considered related to the gavage procedure:
Female No. 65 was sacrificed in extremis for animal welfare reasons on Day 25 post-coitum, as it was noted with labored breathing immediately after dosing and red liquid was noted on the gavage tube . At necropsy, red watery content in the thoracic cavity and dark red foci on the lungs were noted, and the lungs failed to collapse. Furthermore, a small gall bladder and spleen were observed. No microscopic findings were noted for the gastrointestinal tract. This animal was pregnant and had nine live fetuses in utero.
Female No. 64 was sacrificed in extremis on Day 26 post-coitum. Several attempts were made to dose the animal on this day, but this was without success due to repeated coughing1. After removal of the gavage tube, red liquid was found on the dosing tube1 and therefore it was decided to terminate the dosing procedure. Several minutes later1, the animal was noted with labored breathing following which it was decided to euthanize this female for humane reasons. At necropsy, dark red foci on the lungs and abnormal red watery content in the trachea were noted, and the lungs failed to collapse. Other macroscopic findings were dark red discoloration and thickened wall of the colon and no microscopic findings were noted for the gastrointestinal tract. This animal was pregnant and had six live fetuses and one early resorption in utero at necropsy
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight, body weight gain and weight gain adjusted for gravid uterus were considered to be unaffected at 50 mg/kg/day.

At 100 mg/kg/day, body weight loss (-3 g) was observed over Days 7-9 post-coitum followed by comparable body weight gain for the rest of the treatment period. Terminal body weight (Day 29 post-coitum) and gravid uterus adjusted body weight gain comparable to control were noted.

At 200 mg/kg/day, body weight loss was noted over Days 7-15 post-coitum (up to -33 g over Days 9-12 post-coitum), followed by body weight gain comparable to control for the remining treatment period. Lower overall body weight gain (102 vs. 299 g in control) and lower gravid uterus adjusted body weight gain (-363 vs. -224 g in control) were noted.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No test material-related change in food consumption was noted at 50 mg/kg/day.

At 100 mg/kg/day, food consumption was lower (down to 15% of control over Days 12-18 post-coitum; not statistically significant) during the first two weeks of dosing (Days 7-21 post-coitum), followed by normal food consumption thereafter. Overall food intake during the dosing period was 9% lower than control.

At 200 mg/kg/day, food consumption was lower throughout the entire treatment period (not statistically significant between Days 21-29 post-coitum). The largest difference was observed over Days 12-15 post-coitum when food intake was 62% lower than control. Overall food intake during the dosing period was 32% lower than control, an extent which is considered to be adverse.
The slightly lower food consumption at 50 mg/kg/day over Days 7-9 post-coitum (9% below control; not statistically significant) was mainly attributed to the (almost) absent food intake of Female No. 67 over Days 7-8 post-coitum. A possible relation to treatment with the test material could be excluded as this female had (almost) absent food consumption already during the acclimatization period (Days 3-7 post-coitum). Food consumption over Days 9-29 post-coitum and overall food intake during the dosing period were comparable for the low dose and control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No macroscopic observations at scheduled necropsy at doses up to 100 mg/kg/day.

In Group 4 (200 mg/kg/day), 5/21 animals had macroscopic findings in the pyloric region of the stomach consisting of either dark red foci (2 animals) or depressed foci (3 animals), which were considered to be test material-related.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rabbits of this age and strain and were considered common background findings in the rabbit. For several animals (2/22, 2/20 and 3/21 females of the control, 100 and 200 mg/kg/day groups, respectively) the colon was noted to be thick and discolored red. The macroscopic description and microscopic appearance were consistent with a normal anatomic structure in the rabbit colon (fusus coli).
For further details on the results of the pathological examination see the attached summary results tables.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Stomach and the intestinal tract were examined microscopically from control animals (Group 1) and animals dosed at 200 mg/kg/day (Group 4).
Minimal dilatation of the glands was noted in cardiac region of the stomach in 5/21 animals at 200 mg/kg/day.
Minimal or mild erosions (Female Nos. 82 and 87) and/or minimal multifocal acute mucosal hemorrhages (Female No. 87) were noted in the pyloric region of the stomach in 2/21 females correlating to the macroscopic observation of red foci and depressed foci. There were no microscopic correlates for the other animals with macroscopic findings (red foci, area depressed) in the pyloric region.
There were no other test material-related histologic changes in the examined tissues. Remaining histologic changes were considered to be incidental findings. Minimal (rarely mild) erosions in the mucosa of the stomach at the cardio-esophageal junction were common, including control animals, consistent with the literature on background findings in the rabbit. There was no test material related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Postimplantation loss was higher for all test material-treated groups (3.04 in control vs. 8.15, 9.55 and 7.83% at 50, 100 and 200 mg/kg/day). The value for control group was at the lower end of the available historical control data . As no dose response was observed in the treated groups and all values were within the range of the historical control data, the higher postimplantation loss for the test material-treated groups was considered not a result of treatment with the test material.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
At scheduled necropsy, all females were found to be pregnant, except for Female Nos. 1, 12 and 13 (control), 45 and 62 (100 mg/kg/day), and 86 (200 mg/kg/day). Moreover, two females in the 100 mg/kg/day group and one female in the 200 mg/kg/day group had to be terminated preterm.

The number of females with viable litters available for evaluation was 19, 22, 18 and 20 females in the control, 50, 100 and 200 mg/kg/day groups, respectively.
Details on maternal toxic effects:
see attached summary tables for
- clinical observations
- body weights
- body weight gains
- gravid uterus adjusted body weights
- food consumption
- macroscopic pathology
- microscopic pathology
- maternal performance
- Ovarian and uterine examinations and litter observations
- fetal abnormalities by finding
- fetal abnormalities by classification
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
food consumption and compound intake
mortality
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
No test material-related effects on fetal body weights (both sexes) up to 100 mg/kg/day.

At 200 mg/kg/day, mean fetal body weights (male, female and all) were 10-11% lower than control (not statistically significant for females). Mean values for male, female and all weights were below the available historical control data range.
For further details on the results of the fetal examination see the attached summary results tables.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No test material-related external malformations or variations were recorded.

In this study, four fetuses from the low-dose group and three high-dose fetuses presented with external malformations. At 200 mg/kg/day, two littermates (Nos. 84-L7 and -R8) both had cleft palate and abnormalities of hindlimbs and forepaws (malrotation, hyperflexion). The limb and paw bones appeared unaffected on skeletal examination, but cranial findings matched the cleft palate. The four malformed 50 mg/kg/day fetuses were also littermates (Nos. 35-L1, -L2, -R9 and -R11) with limb malformations (hyperflexed hindlimbs) that were not caused by a skeletal abnormality. The occurrences of above findings were in single litters of two different groups and this suggested a genetic cause rather than an effect of the test material. Therefore, a test material-relationship was ruled out. The third externally malformed 200 mg/kg/day fetus (No. 73-R7) had an omphalocele, which is not uncommon among historical control fetuses and was considered a chance finding.

There were no observed external variations or incidental findings.

For further details on the results of the external examination see the attached summary results tables.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No test material-related skeletal malformations or variations were recorded.

In this study, skeletal malformations were observed in 1 (1), 2 (2), 0 (0) and 6 (4) fetuses (litters), from the control, 50, 100 and 200 mg/kg/day groups, respectively.
The two littermates from the high-dose group with external and visceral malformations (Nos. 84-L7 and -R8) had associated skeletal malformations for the cleft palate (palatine split) and one of these (No. 84-R8) also had fused skull bones and sternoschisis. Three other fetuses at 200 mg/kg/day had vertebral anomalies affecting the cervical, thoracic and/or lumbar region and one fetus presented with severely fused sternebrae. The control and two low-dose fetuses also presented with vertebral anomalies (cervical or lumbar region). Vertebral and sternal anomalies are not uncommon among historical control fetuses and due the low incidences and group distribution of these findings, they were not deemed an effect of treatment with the test material.

Regarding skeletal variations it was noteworthy that more cases of unossified hindpaw phalanges were observed at 200 mg/kg/day than when compared to the control and other treated groups. The increase in incidence was not statistically significant and remained within the range of historical control data. Furthermore, the weights of all but one of the eight affected high-dose fetuses ranged from 19.5 to 31.2 grams which was below the group mean fetal weight and significantly below the control group mean fetal weight. Therefore, this was considered a fetal weight-effect and not a direct consequence of treatment with the test material.
Other skeletal variations affected the skull, hindlimb, pelvic girdle, (supernumerary) rib, sternebra and vertebra. These were either scored only in the control group, infrequently, were noted at incidences comparable to the control group and/or occurred in the absence of a dose-relationship. Therefore, they were considered not to be related to treatment with the test-material.
For further details on the results of the skeletal examination see the attached summary results tables.
Visceral malformations:
no effects observed
Description (incidence and severity):
No test material-related visceral malformations or variations were recorded.

Visceral malformations were observed in 1 (1), 3 (3), 2 (2) and 7 (6) fetuses (litters) from the control, 50, 100 and 200 mg/kg/day groups, respectively.
The two littermates at 200 mg/kg/day with cleft palate and limb abnormalities (Nos. 84-L7 and -R8) also had severe dilatation of lateral brain ventricles, and in addition, one fetus (No. 84-R8) had fused and malpositioned kidneys. As stated above, malformations of these fetuses were considered to have a genetic cause and were not attributed to test material-treatment.
The other three high-dose fetuses presented with a variety of blood vessel anomalies. These were retroesophageal aortic arch, tetralogy of Fallot and malpositioned posterior vena cava. All these malformations occurred individually in fetuses from different litters and as all but one (malpositioned posterior vena cava) were listed in historical control data, they were considered not related to test material-treatment.
In the low- and mid-dose groups, malformations concerned the truncus arteriosus, heart, eye, kidney and adrenal gland. The anomalies occurred in single fetuses and due the diverse nature and group distribution an effect of treatment with the test material was ruled out.
The control fetus had a malpositioned kidney, which was considered to be spontaneous in origin.

Visceral variations in this study were observed across all groups in a wide range of structures including gallbladder, abdomen, gonads, innominate artery, intestine, liver, lung, spleen, kidney and ureter. In all cases, the low incidences and group distribution of these findings did not indicate any effect of treatment with the test material.
Incidental findings (not otherwise classified as malformation or variation) only involved a single case of pale spleen in a high-dose fetus which was deemed to be a chance discovery.
For further details on the results of the visceral examination see the attached summary results tables.
Details on embryotoxic / teratogenic effects:
The total number of external, visceral and skeletal malformation observed were within the incidences of the historical controls (see attached documents "20342615_summary-malformations-individual-description" and "20342615_HC-Data_Malformations").
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in litter size and weights
Abnormalities:
no effects observed
Developmental effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
presumably yes

see also attached summary tables (20342615_Summary Results Tables) for


- clinical observations


- body weights


- body weight gains


- gravid uterus adjusted body weights


- food consumption


- macroscopic pathology


- microscopic pathology


- maternal performance


- ovarian and uterine examinations and litter observations


- fetal abnormalities by finding


- fetal abnormalities by classification


 

Conclusions:
Based on the results of this prenatal developmental toxicity study in time mated female New Zealand White rabbits the following maternal and developmental No Observed Adverse Effect Levels (NOAELs) for N,N-Dimethylbenzylamine were established:
Maternal NOAEL: 100 mg/kg/day (based on mortality and low food consumption at 200 mg/kg/day).
Developmental NOAEL: 100 mg/kg/day (based on lower fetal weights at 200 mg/kg/day).
It should be noted that developmental effects in this study were observed at a maternal toxic dose.
Executive summary:

The objectives of this study were to determine the potential of N,N-Dimethylbenzylamine to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits (n=22 per dose group) from Days 7 to 28 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.


The dose levels in this study were selected to be 0, 50, 100 and 200 mg/kg/day, based on the results of the Dose Range Finder (Test Facility Study No. 20342602).


Chemical analyses of formulations were conducted once during the study and confirmed that formulations of test material in 1% Carboxymethyl cellulose + 0.1% Tween80 were prepared accurately and homogenously.


The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, macroscopic examination, and uterine contents (including corpora lutea, implantation sites, pre- and postimplantation loss and number of live and dead fetuses).


In addition, the following parameters were determined for the F1-generation: fetal body weights, sex ratio, external, visceral and skeletal malformations and developmental variations.


In total, three females did not survive until scheduled necropsy.
One female at 200 mg/kg/day was sacrificed in extremis for humane reasons on Day 18 post‑coitum based on prolonged (nearly) absent food consumption. The other two deaths occurred in the 100 mg/kg/day group and were considered related to the gavage procedure.


At 100 mg/kg/day, body weight loss was noted over Days 7-9 post-coitum and lower food consumption was observed over Days 7-21 post-coitum. Both findings were considered non-adverse at this dose level.


At 200 mg/kg/day, increased respiratory rate and slight tremors were noted in one female each on a single day towards the end of the treatment period. Based on similar data from a previous dose range finder study (Test Facility No. 20342602), this was considered test material-related but in the context of the current study non-adverse. Furthermore, non-adverse body weight loss over Days 7-15 post-coitum and non-adverse lower gravid uterus adjusted body weight gain were noted. The test material related lower food consumption was noted during the entire dosing period, with most prominent difference over Days 12-15 post-coitum was considered adverse at this dose level. In several females at 200 mg/kg/day, macroscopic and microscopic findings in the stomach were observed. These included red foci or depressed foci in the pyloric region, partially correlated to minimal mucosal haemorrhages or minimal to mild mucosal erosions microscopically, and minimal dilatation of the gastric glands in the cardiac region. Given the low incidence and severity these findings, they were considered to be non-adverse.


No test material-related changes were noted in any of the remaining maternal parameters investigated in this study (i.e., corpora lutea, uterine contents including implantation sites and pre- and postimplantation loss).

Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study under GLP conditions
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: young-adult (no further data)
- Weight at study initiation: 197-318 g
- Housing: individually
- Diet ad libitum
- Water ad libitum
- Acclimation period: 1 day

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 30 to 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
The test item was administered by gavage to three groups of twenty-four time mated rats between days 5 and 19 of gestation . A further group of twenty-fourtime mated females was exposed to the vehicle only to serve as a control. All females were terminated on day 20 of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item formulations were analyzed for concentration, stability (days 1, 4,and 10) and homogenicity by the test facility
Details on mating procedure:
no data
Duration of treatment / exposure:
gestation day 5 to gestation day 19 (14 days)
Frequency of treatment:
once daily
Duration of test:
gestation day 5 to gestation day 20
No. of animals per sex per dose:
24 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection is based on the results of the preliminary study:
8 time mated females received 0, 50, 100 and 200 mg/kg bw/day test item diluted in arachis oil between gestation day 5 and day 19. Due to early termination of one female , clinical signs of toxicity and actual body weight loss the highest dose was reduced to 150 mg/kg bw on day 5 or day 6 of gestation. The NOAEL in this preliminary study was considered to be 100 mg/kg bw/day.
For the main developmental study a dosage sequence of 0, 35, 75 and 150 mg/kg bw/dy was recommended.
Maternal examinations:
mortality: twice daily
clinical observations: once daily
body weight determination on day 3, 5, 6, 7, 8, 11, 14, 17, and 20 of gestation
food consuption determination on day 3, 5, 8, 11, 14, 17 and 20 of gestation
water consumption daily inspected
Ovaries and uterine content:
uterine / implantation data:
pregnancy status, number of corpora lutea, gravid uterus weight
number, status and intra-uterine position of implantations
Fetal examinations:
external foetal abnormalities, foetal weight foetal sex, placental weight, skeletal and visceral foetal abnormalities
Statistics:
parametric analysis of variance followed by pairwise comparison or non parametirc methods were used
Indices:
pre-implantation loss, post-implantation loss, % male foetuses for determination of sex ratio, fertility index, gestation index
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
----Mortality
There were no unsheduled deaths
----Clinical signs:
from 35 mg/kg bw/day unspecific signs:
-some females showed increased salivation from day 13 earliest
-some females showed noisy respiration or decreased respiration
----Body weight
--at 35 and 75 mg/kg bw/day:
Body weight development was comparable to control animals
-- at 150 mg/kg bw/day
Females showed a marked reduction in body weight gain throughout the the treatment period
Compared to the control animals body weight gain was reduced by 41 %
----Food consumption
at 150 mg/kg bw/day
females showed a reduction (-13 %) in overall food consumption
----Water consumption
No adverse effects were detected
----Post mortem studies
No treatment related macroscopic abnormalities were detected.


----Fertility index and gestation index
was about 100 % in all dose groups including controls
----Number of corpora lutea and gravid uterus weight
at 150 mg/kg bw/day significantly reduced .
Group mean litter value:
number of corpora lutea
at 150 mg/kg bw/day: 12.2 (p<0.01); at 75 mg/kg bw/day: 14.5; at 35 mg/kg/bw/day: 14.9 versus control: 14.7
This intergroup difference was considered to be incidental and unrelatated to treatment due to ovulation and mating occuring prior to the administration of the test substance
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day
Basis for effect level:
other: other:
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No treatment related effects were detected in
----foetal viability (male and female), or in
----foetal weights (male and females), or in
----growth and development.
Sex ratio was in the normal range

Fetal examination:
No treatment related effects were detected on fetal external findings.
No treatment-related effects were detected on skeletal development (including osseous and cartilaginous tissue) amd
none in the type and incidence of skeletal or visceral findings in fetuses from females treated with 150, 75 or 35 mg/kg bw/day.

A teratogenic effect of treatment was not evident up to and including the highest dose level (150 mg/kg bw/day)
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: other:
Abnormalities:
no effects observed
Developmental effects observed:
not specified
Conclusions:
In conclusion, despite general toxicity (body weight reduction) in female rats at 150 mg/kg bw/day there is no evidence that the substance shows developmental toxicity.
Executive summary:

According to OECD TG 414 and under GLP conditions pregnant female rats received orally 0, 35, 75 and 150 mg/kg bw/day by gavage on gestation day 5 -19. All females were terminated on gestation day 20 and cesarian section was performed to evaluate the developing organism.

At the highest dose of 150 mg/kg bw/day females gained markedly less body weight (by 41 % compared to control) and a reduction (-13 %) in overall food consumption. Fertility index and gestation index was calculated to be 100 % in all treated animals including control animals. Post mortem, no macroscopic abnormalities were observed. Thus, due to the reduced weight gain at 150 mg/kg bw/day the NOAEL (maternal toxicity) is considered 75 mg/kg bw/day.

No treatment related effects were detected in foetal viability (male and female), or in foetal weights (male and females), or in growth and development. Sex ratio was in the normal range. No treatment related effects were detected on fetal external findings.

No treatment-related effects were detected on skeletal development (including osseous and cartilaginous tissue) and none in the type and incidence of skeletal or visceral findings in fetuses from females treated with 150, 75 or 35 mg/kg bw/day.

A teratogenic effect of treatment was not evident up to and including the highest dose level (150 mg/kg bw/day)

Thus, the NOEL (No Adverse Effect Level) for reproductive and developmental toxicity isconsidered to be 150 mg/kg bw/day

In conclusion, despite general toxicity (body weight reduction) in female rats at 150 mg/kg bw/day there is no evidence that the substance shows developmental toxicity.

Data source

Materials and methods

Results and discussion

Applicant's summary and conclusion