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Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study under GLP
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
sub-chronic toxicity: oral
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
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
other: EPA OPP 86-5
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Han Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Age of animals at initation of the toxicity phase of the study: 4 weeks
Route of administration:
oral: feed
Details on route of administration:
Dietary
Details on oral exposure:
Groups of 20 male and 20 female Han Wistar rats received Citroflex A-4 via the diet at target
dosages of 100, 300 or 1000 mg/kg/day for 13 weeks. A similarly constituted Control group received
untreated diet. A further 10 male and 10 female rats were assigned to the Control and high dosage
groups for a four-week recovery period following the 13-week treatment period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Group mean achieved dosages over the 13 week treatment period at 100, 300 or 1000 mg/kg/day were
103, 306 and 1013 mg/kg/day for males and 102, 306 and 1024 mg/kg/day for females.
Duration of treatment / exposure:
13 wks for repeated dose toxicity. A 4-week recovery period was a design feature, during which high dose animals received no exposure of test material.
Frequency of treatment:
daily in feed
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Actual: 103 mg/kg bw/d in males, 102 mg/kg bw/d in females.
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Actual: 306 mg/kg bw/d in males, 306 mg/kg bw/d in females,
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Actual: 1013 mg/kg bw/d in males, 1034 mg/kg bw/d in females
No. of animals per sex per dose:
20. 10 additional male and female rats were assigned to the Control and high dose groups for a 4-week recovery period following the 13 week treatment period.
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
During the Toxicity phase of the study the animals were observed daily for general clinical signs and
a detailed physical examination was performed weekly. Bodyweights and food consumption were
recorded weekly. Sexual maturation was assessed in Weeks 4 and 5 and oestrous cycles were
assessed in Week 9. Functional observational battery tests were performed weekly throughout the
treatment period. Ophthalmoscopy examinations were performed during Week 1 and Week 13.
treatment and during Week 4 of recovery.
On completion of the treatment or recovery period, as appropriate, animals were killed and examined
macroscopically; selected organs were weighed and tissues were processed for microscopic
evaluation. Sperm samples were analysed for motility and morphology. Liver samples were removed
from five males and five females in each group and evaluated for peroxisome proliferation.
Statistics:
For organ weights and body weight changes, homogeneity of variance was tested using Bartlett’s test followed by Behrens-fisher test or Dunnett’s test as appropriate. Macroscopic pathology and histopathology data were assessed using Fisher’s Exact test. Estrus cycles were analyzed using the Cochran-Armitage trend test. Other statistical tests used as appropriate were: Williams’ test for a dose-related response; Student’s t-test; Shirley’s non-parametric test for a dose-related response; Steel’s test; and Wilcoxon rank sum test. Significance level was p<0.05..
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Yellow staining of the perigenital and perianal areas of high dose females
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Slighlty low body weight gain for males and females of high dose group.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Slightly high haematocrit, haemoglobin concentrations and erythrocyte counts in males receiving 100, 300 or 1000 mg/kg/day, and slightly
high erythrocyte counts in females receiving 300 or 1000 mg/kg/day. Not toxicologically significant.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Elevations in urea, glucose, sodium potassium, chloride; slightly high albumin and albumin:gobulin ratios; low calcium and phosphorus and total protein concentrations, at various doses in males and females. These biochemical values were within historical control, data ranges, except for the sodium values for males receiving 300 or 1000 mg/kg/day.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Low urinary pH and high urinary protein concentrations at 13 weeks in males receiving 1000 mg/kg/day. These changes were within historical control data ranges and were not apparent at the end of the Recovery period.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of treatment, high bodyweight-relative liver weights were recorded for males and females that received 1000 mg/kg/day. This resolved on completion of the Recovery period.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver peroxisome proliferation after 13 weeks with a statistically significant increases in mean palmitoyl CoA oxidase activity in mid dose males and in high dose males and females, with a statistically significant increase in the mean supernatant protein concentration. Hepatocyte hypertrophy was recorded in the livers of high dose male and female rats.
Other effects:
no effects observed
Description (incidence and severity):
No statistically significant effects on sperm motility, sperm counts or morphology.
Details on results:
Haematological investigations in Week 13 indicated slightly high haematocrit, haemoglobin
concentrations and erythrocyte counts in males receiving 100, 300 or 1000 mg/kg/day, and slightly
high erythrocyte counts in females receiving 300 or 1000 mg/kg/day. These inter-group differences
are not considered to be toxicologically significant, were within historical control data ranges for
these parameters at this laboratory and were not apparent at the end of the Recovery period.

Biochemical examination of the blood plasma in Week 13 indicated high urea concentrations in males
receiving 300 mg/kg/day and in males and females receiving 1000 mg/kg/day, high glucose
concentrations in females receiving 1000 mg/kg/day, high sodium concentrations in males receiving
100, 300 or 1000 mg/kg/day, high potassium concentrations in males receiving 1000 mg/kg/day, high
chloride concentrations in males and females receiving 300 or 1000 mg/kg/day and low calcium and
phosphorus concentrations in females receiving 1000 mg/kg/day. In addition, slightly high albumin
concentrations, with corresponding increases in the albumin to globulin ratio, were recorded in males
receiving 300 or 1000 mg/kg/day, and slightly low total protein concentrations, also resulting in a
corresponding increase in the albumin to globulin ratio, were recorded for females receiving
1000 mg/kg/day. High urea concentrations were still apparent at the end of the recovery period in
females that had previously received 1000 mg/kg/day whilst all other inter-group differences showed
full recovery. The biochemical values of the blood plasma noted above were within historical control
data ranges, except for the sodium values for males receiving 300 or 1000 mg/kg/day.

Urinalysis investigations revealed low urinary pH and high urinary protein concentrations in Week 13
in males receiving 1000 mg/kg/day. These changes were within historical control data ranges and
were not apparent at the end of the Recovery period.

Liver peroxisome evaluation after 13 weeks of treatment indicated statistically significant increases in
mean palmitoyl CoA oxidase activity in males given 300 mg/kg/day and in males and females given
1000 mg/kg/day. Statistically significant increases in the mean supernatant protein concentrations
were also reported in these same dose groups.

Hepatocyte hypertrophy was recorded in the liver of rats receiving 1000 mg/kg/day. In males this
finding tended to occur in the centrilobular region whilst in females it tended to be more generalised.
Hepatocytic hypertrophy was not evident at the end of the Recovery period.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Conclusions:
A 13-week dietary toxicity study was undertaken in weanling F1-generation Han Wistar male and female rats which had been exposed to the test material during gestation and weaning (for approximately 6 weeks). The parental generation of these rats (F0) had been exposed to this substance for 4 weeks prior to mating and 1 week during cohabitation. A 4 week recovery period was included after the 13 weeks of exposure of adult animals (F1). Doses were 100, 300 and 1000 mg/kg bw/d. No significant clinical signs or gross pathology was observed in males or females. Non-specific toxicity was obsrved in high dose animals characteristic of adaptive changes of liver and kidney. These included weak peroxisome proliferation (not relevant to humans), hepatocyte hypertrophy, and alterations in kidney function as seen in clinical chemistry and urinalysis parameters. The NOEL for these potentially non-adverse findings was 100 mg/kg bw/d for males and 300 mg/kg bw/d for females. The overall NOAEL was 1000 mg/kg bw/d for both sexes.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD 408 modified
Qualifier:
equivalent or similar to guideline
Guideline:
other: US EPA OPPTS 870.3100 modified
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
no analysis of uterine contents GD 20
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD 433 EOGRTS
Version / remarks:
without extentions
Principles of method if other than guideline:
Four week repeated dose exposure to adult rats prior to mating, with gravid females exposed during gestation and weaning (6 weeks).
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tributyl O-acetylcitrate
EC Number:
201-067-0
EC Name:
Tributyl O-acetylcitrate
Cas Number:
77-90-7
Molecular formula:
C20H34O8
IUPAC Name:
tributyl 2-acetoxypropane-1,2,3-tricarboxylate
Test material form:
liquid
Remarks:
clear, oily
Details on test material:
Acetyltributyl citrate, batch # 0000016315, Expiry date: January 2002

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Wistar Han rats

Administration / exposure

Route of administration:
oral: feed
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verification occurred; doses were not lower than target doses.
Details on mating procedure:
Males and females cohabitated and mated (1 week).
Duration of treatment / exposure:
P males and females were treated for four weeks prior to mating until scheduled sacrifice. They cohabitated and mated (1 week). After mating, males were sacrificed and toxicity was assessed . Females (P) were placed in separate cages and allowed to deliver, nurse and wean offspring, and then sacrificed. The F1 offspring were placed on test diets for 13 weeks. after which aa recovery groups was test material free for 4 weeks.
Frequency of treatment:
daily in feed
Duration of test:
24 weeks (EOG) plus 4 weeks recovery
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, plain diet
Details on study design:
At the completion of the in utero phase, F1 rats that had been exposed to test material from conception through gestation and continuously from the time of birth were selected (20 unrelated males and 20 unrelated females per dose group for the main study; and 10 unrelated males and 10 unrelated females for the control and high dose recovery groups) and transferred to the 13-week study.

Examinations

Maternal examinations:
Parental animals were evaluated for reproductive endpoints (mating performance, fertility, gestation length and parturition, litter size, numbers of implantations, survival and growth), and F1 animals were evaluated for sexual maturation (balano-preputial separation, vaginal opening, anogenital distance, retained areolae in males, sperm assessments), estrous cyclicity, physical appearance, ophthalmologic effects, neurobehavioral effects, growth, food consumption, survival, hematology, blood chemistry, urinalysis, peroxisome proliferation, organ weights, gross pathology and histopathology. A full range of tissues were retained for theP males and females and reproductive organ tissues were retained for F1 males and females. Microscopic examinations were performed on a standard set of tissues for P males and females, as well as tissues found to be abnormal at necropsy.
Ovaries and uterine content:
Animals allowed to deliver litters naturally.
Fetal examinations:
F1 animals were evaluated for sexual maturation (balano-preputial separation, vaginal opening, anogenital distance, retained areolae in males, sperm assessments), estrous cyclicity, physical appearance, ophthalmologic effects, neurobehavioral effects, growth, food consumption, survival, hematology, blood chemistry, urinalysis, peroxisome proliferation, organ weights, gross pathology and histopathology. A full range of tissues were retained for the Pyes males and females and reproductive organ tissues were retained for F1 males and females.
Statistics:
For organ weights and body weight changes, homogeneity of variance was tested using Bartlett’s test followed by Behrens-fisher test or Dunnett’s test as appropriate. Macroscopic pathology and histopathology data were assessed using Fisher’s Exact test. Estrus cycles were analyzed using the Cochran-Armitage trend test. Other statistical tests used as appropriate were: Williams’ test for a dose-related response; Student’s t-test; Shirley’s non-parametric test for a dose-related response; Steel’s test; and Wilcoxon rank sum test. Significance level was p<0.05.
Historical control data:
yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Yellow staining of the perigenital and perianal areas of high dose females
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
Maternal toxic effects:no effects. There was no effect of treatment upon the F1 generation before they were assigned to the Toxicity phase of this study at approximately four weeks of age.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
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
Other effects:
no effects observed
Description (incidence and severity):
Oestrous cycles, mating performance and fertility were unaffected by four weeks of exposure to tet material at dietary concentrations targeted to generate an intake of 1000 mg/kg/day.
Details on maternal toxic effects:
Maternal toxic effects:no effects. There was no effect of treatment upon the
F1 generation before they were assigned to the Toxicity phase of this study at approximately four
weeks of age.

Details on maternal toxic effects:
Estrous cycles, mating performance, fertility, gestation length and parturition, were all unaffected by treatment. Litter size, survival and growth were similar in all groups and within expected historical control ranges. Although numbers of implantations and litter size at 1000 mg/kg/day were marginally lower than concurrent control group levels, they were within the laboratory’s historical control ranges.

After 13 weeks of treatment, high bodyweight-relative liver weights were recorded for males and females that received 1000 mg/kg/day. This resolved on completion of the Recovery period.

Anogenital distance in both sexes and retention of areolar regions in male offspring were unaffected
by treatment.

There were no macroscopic findings at necropsy of the F0 adults or surplus F1 offspring that were
attributable to treatment.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no effects

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
not examined
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
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Anogenital distance and sexual maturation in both sexes and retention of areolae in male offspring were unaffected by treatment. There were no adverse effects on sperm motility, counts or morphology. There were no findings at necropsy of surplus offspring that were considered to be treatment-related.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
A dietary toxicity study was undertaken in weanling F1-generation Han Wistar male and female rats which had been exposed to the test material during gestation and weaning. The parental generation of these rats (P) had been exposed to this substance for 4 weeks prior to mating and 1 week during cohabitation. Doses were 100, 300 and 1000 mg/kg bw/d. There were no adverse effects observed in reproductive indices or in gross and histopathology in the P generation. There were no significant effects on sperm development or oestrus cycles. There were no adverse effects observed in gestation length, parturition, litter size, survival or growth of offspring. Necropsies of surplus offspring showed no adverse effects. The NOAEL for reproduction and for developmental toxicity was 1000 mg/kg bw/d. Data can be read-across between the structural analogues based on common breakdown products. This is adequate to fulfill the information requirements, to be the basis for classification and labelling decisions, and for risk assessment.