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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 November - 19 December 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Clear slightly viscous amber liquid
Details on test material:
- Name of test material (as cited in study report): Fatty acids, C18 unsat, reaction products with diethylenetriamine
- Substance type: Clear slightly viscous amber liquid
- Physical state: liquid
- Lot/batch No.: S000922
- Expiration date of the lot/batch: 02 July 2017
- Storage condition of test material: At room temperature in the dark under nitrogen
- Purity/composition correction factor required: No
- Hygroscopic: Not indicated
- Volatile: No
- Reactivity: Reactive to oxygen
- Test substance handling: Flush container with nitrogen after handling
- Specific Gravity / Density: 0.926
- pH: 10-12 at concentration of 75%
- Stability in Propylene glycol: Stability for at least 6 hours at room temperature is confirmed over the concentration range 2 to 20 mg/mL, (WIL Research Europe B.V. project 491556) and 2 to 30 mg/mL (WIL Research Europe B.V. project 503870) Stability for at least 8 days in the refrigerator under nitrogen is confirmed over the concentration range 2 to 20 mg/mL, WIL Research Europe B.V. project 503866
- Solubility in Propylene glycol: Up to 200 mg/g

Test animals

Species:
rat
Strain:
other: Wistar (Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Sex: females, nulliparous, nonpregnant and untreated fenimals were used at initiation of the study. (Stock male rats Crl:WI(Han) (outbred, SPF-Quality) were used for mating with the females. After mating these males were placed back in their stock and might be used in further studies.)
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (214 grams).
- Fasting period before study: no
- Housing:
Acclimatization: Animals were housed in groups of 5 animals/cage in Macrolon cages.
Mating: Females were caged together with stock males on a one-to-one-basis or two-to-one-basis in Macrolon cages.
Post-coitum: Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment/nesting material were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

Environmental controls for the animal room were set to maintain 18 to 24°C (range of actual daily mean: 19.6-20.2°C), a relative humidity of 40 to 70% (range of actual daily mean: 48-50%), approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were recorded in the raw data and were considered not to have had any effect on the outcome of the study.

IN-LIFE DATES: From: 18 November - 19 December 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for the specific gravity of the vehicle (1.036) and test substance. No correction was made for the purity/composition of the test substance.
- Storage conditions of formulations: At ambient temperature.

VEHICLE
- Justification for use and choice of vehicle: Based on Project 491556 (combined 28-day repro screening study with same batch of test substance).

DOSE VOLUME:
5 ml/kg body weight. Actual dose volumes were calculated according to the latest body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 8 days in the refrigerator under nitrogen was also determined (highest and lowest concentration). Additional stability measurements over 6 hours were performed on concentrations of 2 and 30 mg/kg. The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Details on mating procedure:
- M/F ratio per cage: 1/1.
- Age at start of mating of the animals in the study: Approximately 12 weeks
- Length of cohabitation: until sufficient mated females had been obtained for each dose group.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage and/or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
Duration of treatment / exposure:
Duration of treatment: From Days 6 to 19 post-coitum, inclusive.
Frequency of treatment:
Once daily for 7 d/w.
Duration of test:
Duration of test: From Day 0 to 20 post-coitum, inclusive.
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: Dose levels were based on a MTD study (NOTOX Project 491555) and a combined 28-day/repro screening study (NOTOX Project 491556). In the MTD study, 3 rats/sex/group were exposed for 10 days at 50 and 250 mg/kg. At 250 mg/kg, toxicity consisted of clinical signs, decreased body weights and food consumption, and decreased weights of the thymus and spleen for one female. In the combined 28-day/repro screening study, 10 rats/sex/group (plus 5 recovery males in the control and high dose group) were exposed at 10, 30 and 100 mg/kg. Histopathology revealed foamy macrophage foci in the ileum of all selected males and females at 100 mg/kg/day, a slightly increased incidence and degree of macrophage foci in the mesenteric lymph node of both sexes at 100 mg/kg/day, and increased incidence of lymphoid atrophy in the thymus of females at 100 mg/kg/day. At the end of the 14-day recovery period for males, foamy macrophage foci in the ileum had completely resolved, whilst macrophage foci in the mesenteric lymph node persisted at higher severity than observed at the end of treatment. Given the persistence of this finding it was considered to be of an adverse nature.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.

BODY WEIGHT
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 17 and 20 post-coitum.

FOOD CONSUMPTION
- Time schedule for examinations: Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum.

WATER CONSUMPTION
No. Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

GENERAL REPRODUCTION DATA
- Mating date and confirmation of pregnancy were recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.

POST-MORTEM EXAMINATIONS
- Sacrifice on Day 20 post-coitumusing an oxygen/carbon dioxide procedure and subsequently subjected to a to an external, thoracic and abdominal examination.
Ovaries and uterine content:
Each ovary and uterine horn of animals surviving to planned necropsy were dissected and examined
as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The weight of each fetus.
- The sex of each fetus from the ano-genital distance (during necropsy) and also from gonadal inspections (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.
Fetal examinations:
External, visceral and skeletal fetal findings were recorded as developmental variations or malformations.
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: Groups 1 and 4
- Head examinations: Yes: half per litter
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Ref. 10) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
For each litter the following calculations were performed:
Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites) / number of corpora lutea x 100
Post-implantation loss (%) = (number of implantation sites - number of live fetuses) / number of implantation sites x 100
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:
Viable fetuses affected / litter (%) = number of viable fetuses affected/litter / number of viable fetuses/litter x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
MORTALITY:
No mortality occurred during the study period.

CLINICAL SIGNS:
At 150 mg/kg, one female (no. 85) showed hunched posture, piloerection and reduced faeces production during the last two days of treatment. This animal also showed affected body weight, food consumption and macroscopic findings, and therefore these clinical signs were regarded treatment related. At 150 mg/kg, eight additional animals also showed clinical signs (rales, piloerection and/or pale faeces). However, as these signs were only noted for 1-3 days, these signs were not considered toxicologically relevant. Incidental findings that were noted for the other groups consisted of black or red discolouration of the tail, rales, alopecia, and pale faeces. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

BODY WEIGHTS:
Reduced body weights and body weight gain from Day 12 post-coitum onwards, and lower body weight gain corrected for gravid uterus weight were noted for approximately half of the animals at 150 mg/kg. Two animals (nos. 85 and 86) were most severely affected; female no. 86 gained only 2% and female no. 85 lost 18% by the end of the treatment period. At 15 and 50 mg/kg, body weights and body weight gain (also body weight gain corrected for uterine weight) remained in the same range as controls over the treatment period.

FOOD CONSUMPTION:
At 150 mg/kg, food consumption was statistically significantly reduced during the complete treatment period (Days 6-20 post-coitum). Food consumption of female nos. 85 and 86 was severely reduced from Day 12 post-coitum onwards. Food consumption before or after allowance for body weight was similar between controls and animals treated at 15 and 50 mg/kg.

MACROSCOPIC EXAMINATION:
At 150 mg/kg, two females (nos. 85 and 86) had the gastro-intestinal tract distended with gas and small thymus and spleen. In addition, one of these also showed irregular surface of the stomach and was emaciated. The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore not considered to be toxicologically relevant, and included several reddish foci on the thymus, uterus containing fluid, alopecia, and pelvic dilation of the kidneys.

MATERNAL PREGNANCY DATA:
There were 22, 21, 21, and 18 pregnant females in the control, 15, 50 and 150 mg/kg groups, respectively, with 22, 21, 21, and 18 litters available for evaluation. No significant differences were observed between control and treated groups regarding the number of corpora lutea, implantation sites, viable or dead fetuses, early or late resorptions, or pre- and post-implantation loss.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
> 50 - < 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FETAL FINDINGS
LITTER SIZE
No treatment related effect on litter size was noted up to 150 mg/kg. The mean number of viable fetuses per litter was 11.3, 11.5, 11.9 and 11.3 in the control, 15, 50 and 150 mg/kg groups, respectively.

SEX RATIO
There were no treatment-related effects on the sex ratio of the fetuses.

FETAL BODY WEIGHT
No toxicologically relevant change was noted for fetal body weights up to 150 mg/kg. One litter at 150 mg/kg showed a very low mean fetal body weight (i.e. 2.0 gram). This was considered secondary to the health status of this dam. Body weights of fetuses (sexes combined) were 3.5, 3.4, 3.5 and 3.3 grams for the control, 15, 50 and 150 mg/kg groups, respectively.

FETAL MORPHOLOGICAL EXAMINATIONS
EXTERNAL MALFORMATIONS AND VARIATIONS
The numbers of fetuses (litters) available for external, visceral and skeletal morphological evaluation were 248(22), 241(21), 249(21) and 204(18) in Groups 1, 2, 3, and 4, respectively. Soft tissue cephalic examination was done for approximately half of the fetuses of all groups and skeletal examinations were done for all the fetuses of Groups 1 and 4. Malformations were observed in 6(5), 0(0)*, 4(3)* and 5(4) fetuses (litters) in these same respective dose groups.
* Note: For Group 2 and 3 treated at 15 and 50 mg/kg, these numbers of malformations were based on external, visceral and soft tissue cephalic examination; no skeletal examination had been performed for these groups.

EXTERNAL MALFORMATIONS AND VARIATIONS
There were no treatment related effects on fetal external morphology up to 150 mg/kg. Two external malformations were noted in this study. One fetus of the control group had a cleft palate (over the entire length) and one fetus at 50 mg/kg had exencephaly (absence of part of the cranial vault, with the brain protruding outside the skull). One external variation (i.e. subcutaneous edema in the neck) was noted for one fetus at 150 mg/kg. At this single occurrence in the control, mid and high dose group, these findings were not considered to be treatment related.

VISCERAL MALFORMATIONS AND VARIATIONS
There were no treatment related effects on fetal visceral morphology up to 150 mg/kg. Visceral malformations were observed in four fetuses (three litters). Internal hydrocephaly was noted for one fetus of the control group and one fetus at 50 mg/kg. At 50 mg/kg, one fetus had a small eye and one fetus showed persistent truncus arteriosus and absent eyes. At this low incidence and without a dose response relationship, these malformations were not considered treatment related. In addition, all findings (except persistent truncus arteriosus) were noted before in historical control fetuses. As persistent truncus arteriousus was noted for only one fetus at the mid dose group, it was considered a chance finding and not related to treatment. Visceral variations noted for fetuses in the control and/or treated group(s) were small supernumerary liver lobe, dilated ureter, liver appendix, partially undescended thymus horns, and convoluted ureter. These variations were not considered to be treatment related, because they occurred infrequently, in the absence of a dose related trend and/or at frequencies within the historical control range.


SKELETAL MALFORMATIONS AND VARIATIONS
There were no treatment related effects on fetal skeletal morphology up to 150 mg/kg. Skeletal malformations were observed in four fetuses (three litters) of the control group and five fetuses (four litters) at 150 mg/kg. One control fetus showed fusion of the sternebrae (nos. 4 and 5 with thread-like ossification bridge) with severely malaligned sternebrae (no. 4; slight no. 3). As this concerned a control fetus, it was not treatment related. Bent limb bones were noted for three control fetuses (two litters) and five high dose fetuses (four litters). One of these high dose fetuses also showed sternoschisis (nos. 1-6). Bent limb bones have been noted in 17 historical control fetuses (15 litters) with a maximum incidence of 2.0%. The litter incidence of the concurrent control group is 1.2% and 3.6% at 150 mg/kg. Even though the incidence at 150 mg/kg is outside the historical control range, these findings were not considered treatment related as the absolute number of affected fetuses at 150 mg/kg was only slightly above the concurrent control group (5 fetuses at 150 mg/kg versus 3 control fetuses). Moreover, bent limb bones are considered reversible (Ref. 12) and are therefore not adverse. In addition, as sternoschisis was noted for a single fetus, and as it was seen previously in the historical control data (2 fetuses), it was considered a chance finding and not toxicologically relevant. Skeletal variations noted for fetuses in the control and/or high dose group were unossified or reduced ossification of several bones (skull, entire sternum, vertebral centra, vertebral arches, pubis, ischium, ribs, sternebrae nos. 1, 2, 3, 4 5 and/or 6, vertebral centra, hyoid, metacarpals and/or metatarsals), 14th full or rudimentary ribs, bent ribs, 7th cervical full or rudimentary ribs, cervical centrum no. 1 ossified, slight or moderate malaligned sternebrae, caudal shift of pelvic girdle, and branched sternebrae. These variations were not considered to be treatment related, because they occurred at similar or higher frequencies in the control group, occurred infrequently and/or occurred at frequencies within the historical control range.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

FORMULATION ANALYSIS:

Accuracy of preparation: The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). A small response at the retention time of the test substance was observed in the chromatograms of the Group 1 formulation. It was not considered to derive from the formulation since a similar response was obtained in the analytical blanks.

Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Stability: Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Applicant's summary and conclusion

Conclusions:
The prenatal developmental toxicity study (OECD 414) on AAI-DETA resulted to a maternal NOAEL of 50 mg/kg. The developmental NOAEL was at least 150 mg/kg.
Executive summary:

Fatty acids, C18 unsat, reaction products with diethylenetriamine (AAI-DETA)was evaluated for developmental toxicity/teratogenicity in a study performed according to theOECD 414 guideline and in compliance to GLP. Eighty-eight mated female Wistar Han rats were assigned to four dose groups. The test item was administered once daily by oral gavage from Days 6 to 19 post-coitum at doses of 15, 50 and 150 mg/kg.

Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on one day during treatment were analyzed for accuracy, homogeneity and stability. On an additional occasion, extra stability measurements were performed.

All animals surviving to Day 20 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparo-hysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative, all fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous ethanol and stained with Alizarin Red S. Skeletal examinations were performed for all fetuses of de control and high dose group..

 

Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

Maternal findings:

Maternal toxicity was noted at 150 mg/kg, and consisted of reduced body weight gain and food consumption. The two dams that were most affected, also showed corroborative macroscopic abnormalities (gastro-intestinal tract distended with gas, small thymus and spleen, irregular surface of the stomach and/or emaciation), and one of these showed clinical signs (hunched posture, piloerection and reduced faeces) during the last two days of treatment. No maternal toxicity was observed in the 15 and 50 mg/kg groups.

Developmental findings:

No developmental toxicity was observed up to the highest dose levels tested (150 mg/kg).

Based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for Fatty acids, C18 unsat, reaction products with diethylenetriamine was established as being 50 mg/kg. The developmental NOAEL was at least 150 mg/kg.