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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

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

Materials and methods

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

Test material

Constituent 1
Reference substance name:
Bis(2-ethylhexyl) adipate
EC Number:
203-090-1
EC Name:
Bis(2-ethylhexyl) adipate
Cas Number:
103-23-1
IUPAC Name:
bis(2-ethylhexyl) adipate
Constituent 2
Reference substance name:
Bis(2-ethylhexyl)adipat
IUPAC Name:
Bis(2-ethylhexyl)adipat
Test material form:
other: liquid
Details on test material:
please refer to confidential details on test material

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: : female albino rabbits, New Zealand White (NZW) strain (SPF-Quality)
- Source:Charles River, Chatillon sur Chalaronne, France
- Age at study initiation: females were approx. 16-19 weeks / adult males
- Weight at study initiation: 3036 - 4937g
- Fasting period before study: no
- Housing: Females were individually housed
- Diet (e.g. ad libitum): ad libitum (pressed hay and wooden sticks)
- Water (e.g. ad libitum):tap-water ad libitum
- Acclimation period: at least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24°C
- Humidity (%): 40-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): once weekly
- Mixing appropriate amounts with (Type of food): Standard powder rabbit diet (Global Diet 2030 from Harlan Teklad, Nucedola, Milanese, Italy
- Storage temperature of food: kept at room temperature, diets were used within 8 days or stored in the freezer at ≤-15°C
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
After termination, the actual test substance intake was estimated based on the body weight and food consumption values.
Concentrations in the diet were verified for all dose groups and were in the range of 90 to 110% of the target concentration.
Homogenicity was verified for low and mid dose preparations (coefficient of variation <= 10%).
Details on mating procedure:
One female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was established by visual observation of mating. This day was designated Day 0 post-coitum.
Duration of treatment / exposure:
from days 6 to 29 post-coitum
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 40, 80 and 160 mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
Group 1, 2, 3 and 4 consisted of 22, 27, 23 and 21 females, respectively.
At termination of the study, 5 animals were found to be non-pregnant in Group 2, which resulted in a low number of animals available for evaluation in this group. Therefore, it was decided by the Sponsor to include another 5 animals in Group 2. The additional animals were originally mated in another prenatal developmental toxicity study from the same Sponsor.
Female no. 48 (Group 3) was euthanized on Day 4 post-coitum (found to have a urinary infection at macroscopic examination). This female was replaced by a spare female.
Animal number 70 was a male, as by mistake after successful mating the male was placed back in the female cage and vice versa; obtained data was excluded from the report but was kept in the raw data.
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were based on a maternal toxicity study in which mated rabbits (5 per group) were exposed from Days 7-29 post-coitum by dietary administration at target dose levels of 0, 100, 300 and 1000 mg/kg bw/day, and one group of animals was dosed by oral gavage at 300 mg/kg bw/day (to investigate a possible palatability problem using dietary exposure). Severe toxicity was noted at 300 mg/kg bw/day (both exposure routes) and 1000 mg/kg bw/day. No toxicity was noted at 100 mg/kg bw/day.
The corresponding mean test article intakes for these dose levels were 105, 240 and 498 mg/kg bw/day, respectively.
Based on the results in this prenatal maternal toxicity study the selected dose levels for the prenatal developmental toxicity study of BIS(2-ETHYLHEXYL)ADIPAT in rabbits were 40, 80, and 160 mg/kg.

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- 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. Cage debris was examined to detect abortion or premature birth

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: Days 0-3, 3-6, and daily from Days 6-29 post-coitum

WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

MORTALITY: At least twice daily
Ovaries and uterine content:
The ovaries and uterine content was examined after termination.
Examinations included:
- 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 (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.
Animals sacrificed before planned necropsy were subjected to relevant examinations of the ovaries and uterine horns.
Fetal examinations:
External:
Each viable fetus was examined in detail and weighed. All live fetuses were euthanised by administration of approximately 0.3 mL (= 60 mg) of sodium pentobarbital (Euthasol® 20%; AST Farma B.V., Oudewater, The Netherlands).
Visceral (Internal):
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar. The sex of all fetuses was determined by internal examination.
The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution (Klinipath, Duiven, The Netherlands). Tissues were then transferred to a 70% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent processing and soft-tissue examination using the Wilson sectioning technique. After examination, the tissues were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter were processed for skeletal examination as described below.
All carcasses, including the carcasses without heads, were eviscerated, skinned and fixed in identified containers containing 96% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent examination of skeletons.
Skeletal:
The eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide (Merck, Darmstadt, Germany) and stained with Alizarin Red S (Klinipath, Duiven, The Netherlands) by a method similar to that described by Dawson. Subsequently, the skeletal examination was done on all fetuses. All specimens were archived in glycerin (Klinipath, Duiven, The Netherlands) with bronopol (Alfa Aesar, Karlsruhe, Germany) as preservative.

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.

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 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. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No maternal toxicity was observed in the 40, 80 and 160 mg/kg bw/day groups. Average compound intake was 36, 70, and 145 mg/kg bw/day.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
160 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
160 mg/kg bw/day (nominal)
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
No developmental toxicity was observed in the 40, 80 and 160 mg/kg bw/day groups. There were no effects on the external, visceral and skeletal fetal morphology up to 160 mg/kg bw/day

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Mortality

No treatment related mortality occurred up to 160 mg/kg bw/day.

There were seven females terminated before planned necropsy:

At 40mg/kg bw/day, one female (no. 31) showed an early delivery on Day 29 post-coitum and one female (no. 35) aborted on Day 27 post-coitum.

At 80mg/kg bw/day, two females aborted (no. 45 on Day 29 post-coitum and no. 61 on Day 26 post-coitum), one female (no. 55) showed an early delivery on Day 27 post-coitum, and one female (no. 48) was terminated on Day 4 post-coitum. Female no. 48 showed marked red urogenital discharge on Day 4 post-coitum. As treatment had not started yet, it was decided to remove this animal from study and replace it by a reserve female. At macroscopic examination, female no. 48 showed several abnormalities of the urinary bladder (gelatinous appearance, altered urinary bladder wall (nodule, foci, thickened, reddish discolouration) and its contents (gray-white, watery-cloudy).

At 160mg/kg bw/day, one female (no. 82) aborted on Day 27 post-coitum.

 

Clinical signs

No treatment related clinical signs were noted up to 160 mg/kg bw/day.

Reduced faeces production was noted for several rabbits of all groups (included the control group). As the incidence was comparable for all groups, this finding was not considered caused by treatment of the test substance.

Incidental findings that were noted included alopecia, scars, scabs, a wound, red discolouration of the urine, diarrhea, and dark or enlarged faeces balls. These findings 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. At the incidence observed, these signs were considered of no toxicological relevance.

 

Body weights

Body weights and (corrected) body weight gain of treated animals up to 160 mg/kg bw/day remained in the same range as controls over the treatment period.

Food consumption

No toxicologically relevant treatment related changes were noted for food consumption before or after allowance for body weight up to 160 mg/kg bw/day.

At 80 mg/kg bw/day, mean (relative) food consumption was slightly lower (not statistically significant) from Day 20 post-coitum onwards, which was due to low consumption of individual animals (nos. 45, 46, 50, 54, 55, 61, and 49). As no dose response relationship was noted, it was not considered toxicologically significant.

The statistically significantly higher values noted on a few occasions were not considered toxicologically relevant as no dose response was noted, and a decrease rather than increase would be expected in the case of toxicity.

 

Test article intake

The mean test article intake values(mg test article/kg body weight/day)are summarized in the following table:

 

 

Dose level approximation

Group 2

40 mg/kg bw/day

Group 3

80 mg/kg bw/day

Group 4

160 mg/kg bw/day

 

 

 

 

Post-coitum days 6-29

36

70

145

 

The mean test article intake was highest during the first days and slowly declined during the treatment period.

It should be noted that a large individual variation in test article intake was noted within each group as a large range in food consumption was noted during treatment. This range varied between 23 and 53 mg/ bw/day for Group 2, between 52 and 77 mg/kg bw/day for Group 3, and between 87 and 187 mg/kg bw/day for Group 4.

  

Macroscopic examination

Macroscopic observations at necropsy did not reveal any compound-related alterations.

  

Maternal pregnancy data

For the control, low, mid and high dose groups, 20, 19, 19 and 19 litters with viable fetuses were available on Day 29 post-coitum.

 

At the control group, two females were not pregnant.

At 40 mg/kg bw/day, six females were not pregnant, one female aborted and one female had an early delivery.

At 80 mg/kg bw/day, three females showed an abortion or early delivery. One female (no. 48) was euthanized early and was replaced by a reserve animal.

At 160 mg/kg bw/day, one female was not pregnant and one aborted.

 

Fetal findings

Litter size

Litter size was unaffected by treatment up to 160 mg/kg bw/day.

Mean number of viable fetuses per litter were 10.7, 8.9, 11.2 and 9.0 in the control, 40, 80 and 160 mg/kg bw/day dose groups, respectively.

The number of early resorptions was slightly higher (total of 10 compared to 4 in the control group), however not statistically significant. As these incidences were within normal limits, it was not considered to be compound-related.

 

Sex ratio

No toxicologically relevant findings were noted for sex ratio for treatment up to 160 mg/kg bw/day.

 

Fetal body weight

No toxicologically relevant findings were noted for fetal body weights with treatment up to 160 mg/kg bw/day.

Mean fetal body weights (both sexes combined) were 39.5, 41.3, 37.0 and 40.1 gram in the control, 40, 80 and 160 mg/kg bw/day dose groups, respectively.

At 80 mg/kg bw/day, slightly lower fetal body weights (not statistically significant) were noted which were mainly caused by four litters (nos. 46, 50, 53 and 54). For all but no. 53, markedly lower maternal food consumption was observed in the does and may be therefore linked to the lower fetal weights. At this low incidence and as the high dose of 160 mg/kg bw/day showed unaffected fetal body weights, it was considered to be incidental.

 

External Malformations and Variations

There were no treatment related effects on external morphology up to 160 mg/kg bw/day.

There were five fetuses with external malformations; all malformations occurred only once and as these findings had been noted in historical control fetuses, it was not considered to be related to treatment.

Findings consisted of:

At 80 mg/kg bw/day, one fetus showed a distended abdomen (confirmed by visceral examination as abdominal ascites, greenish fluid with no apparent visceral origin) and one aborted dead fetus showed omhpalocele (protrusion of several loops of intestine and liver through a defect in the abdominal wall at the umbilicus, not covered by a thin translucent sac; data not shown in summary tables).

At 160 mg/kg bw/day, one fetus showed hyperextension of the right hindlimb (with no apparent skeletal origin), one fetus showed carpal and/or tarsal flexure on the right forepaw (confirmed skeletally as absent right radius) with ectrodactyly (absent first digit right forepaw; confirmed skeletally), and one fetus showed spina bifida (vertebral column open in the sacral region; length of defect 0.8 cm; confirmed at skeletal examination) and a small tail (8 mm in length; confirmed skeletally).

No external developmental variations were observed in any of the fetuses.

 

Visceral Malformations and Variations

There were no treatment related effects on visceral morphology up to 160 mg/kg bw/day.

Visceral malformations included tetralogy of Fallot, absent lung lobe, malpositioned testis, and persistent truncus arteriosus. For these findings no dose response was apparent or they only occurred at the control group. Abnormal lung lobation and internal hydrocephaly were noted for single fetuses at 160 mg/kg bw/day. As these malformations were also noted in the historical control database and they only occurred singly, these findings were not considered treatment related.

Visceral variations noted for fetuses in the control and/or treated group(s) were supernumerary artery or spleen, absent or small gallbladder, liver with appendix, retrocaval ureter, malpositioned left carotid, convoluted ureter, partially undescended thymus horn, constricted spleen, absent renal papilla, bilobed gallbladder, and discoloured liver. These variations were not considered to be treatment related, because they occurred infrequently, in the absence of a dose related trend, at frequencies within the historical control range and/or were seen in control fetuses only.

 

Skeletal Malformations and Variations

There were no treatment related effects on skeletal morphology up to 160 mg/kg bw/day.

Skeletal malformations seen in the control and/or treated groups included split skull bones, anomalies of different bones (skull, sternum, caudal vertebral, rib), fused sternebrae, severely malaligned sternebrae, absent limb bones, and vertebral anomaly with or without associated rib anomaly. All these skeletal malformations were not considered to be treatment related as they were isolated findings, occurred at frequencies within the range of available historical control data and/or were seen in control fetuses only.

Skeletal variations seen in control and/or treated groups included full or rudimentary 13thrib(s), caudal or cranial shift of pelvic girdle, 7thcervical rudimentary or full rib(s), supernumerary site (vertebra, skull), ossification site (sternum) or sternebra, nodulated rib(s), and unossified or reduced ossification of different bones (sternebra(e) nos. 5 and/or 6, metacarpal(s) and/or metatarsals(s), vertebral centra, skull bone line, hyoid body and/or arches, tarsals, pubis). All these skeletal variations were not considered to be treatment related as they occurred at a very low incidence, at frequencies within or just outside the range of available historical control data and/or were seen in fetuses of the control group only.

The statistically significantly lower incidence noted for slightly to moderately malaligned sternebrae at 160 mg/kg bw/day was not considered toxicologically relevant as the incidence was within the historical control data and an increase rather than a decrease would be expected in the case of relevant toxicity.

 

 

 

     

Applicant's summary and conclusion

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