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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:
From May 13, 1991 to April 23, 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Test animals
- Age: Males approx. 63 d; Females: approx. 56 d
- Weight: Males 265 – 320 g upon arrival; Females 172 – 200 g upon arrival
- Fasting period before study: no data given
- Housing: Before mating males were housed singly, females were housed two to a cage.
- During mating 1 male and 1 female were housed individually.
- After mating copulation plug positive females and therefore considered successfully mated
- were housed singly for the duration of the study in stainless steel wire mesh cages.
- Diet: Access ad libitum; Ground Certified Rodent Chow ® (#5002, Ralston Purina Co., St. Louis, MO).
- Water: ad libitum, municipal tap water
- Acclimation period: approx. 2 wks
Environmental Conditions
- Temperature (°C): 19 – 25
- Humidity (%): relative 40 – 70
- Photoperiod: 12 h dark/12 h light; light period 5 – 17 h

In-Life dates: From May 27-30, 1991 to June 14 (gd 15); May 28 – 30, 1991 was gd0.
- Treatment began on gd6 (June 3-5, 1991). Dams were sacrificed on gd21 (June 18 – 20, 1991)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
Preparation of dosing solutions:
Control: Milli-Q ® water, volume according to highest test substance volume selected (1,42 mlL/ kg bw/day)
Dose levels: The dose levels referring to the active ingredient were 100, 300, and 1000 mg/kg bw/day administered undiluted once a day, based on the dam's body weight on gestational day 6, the percent active ingredient (76,6%) and the specific gravity of the test substance (0,92 g/cm3).
Dosage volumes were not changed to account for increases in gestational body weight after gd6. Accordingly the applied test substance volumes were 0,142, 0,426, and 1,42 mg/kg bw/day
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not applicable
Details on mating procedure:
Proof of pregnancy: Females were checked once daily for vaginal copulation plugs in the morning and the paperboard beneath the cages was checked twice daily for dropped copulation plugs. The observation of a vaginal or dropped copulation plug was considered evidence of successful mating. Each male was paired only once in this study.
Duration of treatment / exposure:
10 d, from gd6 through gd15
Frequency of treatment:
Once daily
Duration of test:
22 days (till gd21)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
based on active ingredient (a.i.)
Dose / conc.:
100 mg/kg bw/day
Remarks:
based on active ingredient (a.i)
Dose / conc.:
300 mg/kg bw/day
Remarks:
based on active ingredient (a.i)
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
based on active ingredient (a.i)
No. of animals per sex per dose:
25

Evaluated pregnant rats: group 1-4: 21/23/22/25 (females with viable fetuses)

Animals evaluated for maternal toxicity: group 1-4: 22/23/24/25
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: Based on a former dose range finding study
Rationale for animal assignment (if not random): The rat is a commonly used rodent species for embryotoxic studies.
Group size per dose level: 22/23/23/25 pregnant animals per dose group

Examinations

Maternal examinations:
Cage side observations: yes
Time schedule: daily
Detailed clinical observations: Yes
Time schedule: daily
Clinical signs: Yes
daily observations for behavior, external appearance and general condition
Viability: Yes
daily checks twice daily for morbidity and mortality
Body weight: yes
Time schedule for examinations: Maternal body weights were taken on gd0, gd6 (prior to the onset of dosing), gd9, 12, 15, 18, and 21
Food consumption: yes
Food consumption was measured at three-day intervals throughout the study (gd0 through 21).
Water consumption: no
Post-mortem examinations: Yes
Organs examined: Macroscopic examination of uteri, ovaries, cervix, vagina, peritoneal and thoracic cavities.
Maternal liver and Gravid Uteri weights, Number of Corpora lutea, Number and status of Implantation sites.
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
Fetal examinations:
Fetal examinations
External examinations: yes: all litter
Soft tissue examinations: yes; approx. 50%
Skeletal examinations: yes, 50%
Head examinations: yes
other: Number of fetuses (live and dead), sex and viability of fetuses, variations and malformations, including cleft palate
Statistics:
Levene's test for equality of variances, analysis of variance (ANOVA) and t-tests. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated equal variances, and the ANOVA was significant, a pooled t-test was used for pair wise comparisons. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by a separate variance t-test for pair wise comparisons. Nonparametric data were statistically evaluated using the Kruskal-Wallis test, followed by the Mann-Whiney U test when appropriate. Incidence data were compared using the Fisher's Exact Test. With the exception of analyses for fetal malformation and variation data, all statistical evaluations were performed using BMDP Statistical Software (Dixon, 1990). For all statistical tests, the probability value of <0,05 (two-tailed) was used as the critical level of significance.

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 substance related maternal toxicity detected up to the highest dose tested.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
No embryotoxicity or teratogenicity detected up to the highest dose tested

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
other:
Basis for effect level:
other: embryotoxicity

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:

Mortality:

300 mg/kg bw/day dose group: One female became moribund and was sacrificed on gd10.

Clinical signs:

300 mg/kg bw/day dose group: 2 dams exhibited audible respiration during or subsequent to the treatment period.

One dam exhibited swelling in the face, urogenital area wetness, gasping and perinasal and perioral encrustation. This animal was sacrificed due to her moribund condition on gd10. None of these signs were considered to be test substance related due to their absence in the dose range finding study up to doses of 1875 mg/kg bw/day and the lack of any dose relation in the current study.

1000 mg/kg bw/day dose group: 3 dams exhibited audible respiration during or subsequent to the treatment period.

No treatment-related differences in gestational parameters including total number of implantations, number of viable and nonviable implants in any dose group.

Body weight: No treatment-related effects on gestational body weights and body weight gain, corrected body weight, corrected body weight gain, absolute and relative liver weight, and gravid uterine weight . Fetal body weights per litter were not affected by treatment.

Increased food consumption in the 100 mg/kg bw/day dose group was not considered to be related to treatment due to the lack of a dose-relationship.

NECROPSY FINDINGS IN DAMS AT TERMINATION:

One female in the 300 mg/kg bw/day dose group which was sacrificed due to her moribund condition, had gas-filled intestines, mucoid fluid or material in the nose turbinates, abnormal material in the trachea and discolored and consolidated lungs. One further animal in this dose group had no implants in one uterine horn.

Two females in the 1000 mg/kg bw/day dose group contained blood in the uterus (detected with Hemastix reagent strips) One further female had discolored lungs (dark red) in all lobes.

REPRODUCTION DATA OF DAMS (0, 100, 300, 1000 mg/kg bw):

-         Corpora lutea:338 (16.1 per dam), 380 (16.5 per dam), 364 (15.7 per dam), 387 (15.5 perdam)

-         Implantation sites (Total implants): 340 (15.5 per dam), 379 (16.5 per dam)*, 363 (15.7 perdam)**, 382 (15.3 per dam)

-         Resorptions: 10 (0.5 per dam), 7 (0.4 per dam), 12 (0.5 per dam), 53 (2.7 per dam)**

-         Early resorptions:13 (0.6 per dam), 21 (0.9 per dam), 17 (0.7 per dam), 14 (0.6 per dam)

-         Late resorptions:0 (0 per dam), 1 (0 per dam), 2 (0.1 per dam), 0 (0 per dam)

-         Live (Viable) fetuses:327 (14.9 per dam), 356 (15.5 per dam), 343 (14.3 per dam),367 (14.7 per dam)

-         Malformations (external, skeletal, soft tissue) (fetal incidence): none significantly different from Control

-         Skeletal variations (fetal incidence):

-         Findings: Anterior arch of atlas poorly ossified; Statistical significance of affected litters in 1000mg/kg bw/day dose group;

% affected litters (71.4, 87.0, 86.4 96.0*)

-         - Skeletal variations (fetal incidence):

-         Findings: Majority of forelimbs unossified; Statistical significance of affected litters in 300mg/kg bw/day dose group;

% affected litters (38.1, 26.1, 4.5**, 24.0)

-         - Soft Tissue variations (fetal incidence):

-         Findings: Excessive bleeding at umbilicus; Statistical significance of affected litters in100 mg/kg bw/day dose group;

% affected litters (23.8, 0*, 27.3, 40.0)

-         Other External variations (fetal incidence): none significantly different from control

Weight of fetuses: No treatment related effects on fetal body weights (all fetuses, male or female) observed in any group. The statistically significant* differences in mean male and female body weights in litters at 100 mg/kg bw/day were not considered to be treatment-related due to the lack of a dose-response relationship.

* Significantly different from control, p= 0.05 using two-tailed Fisher's exact test

** Significantly different from control, p= 0.01 using two-tailed Fisher's exact test


Applicant's summary and conclusion

Conclusions:
Under the study conditions, the test substance maternal, emryotoxic and teratogenic toxicity NOELs of the test substance was determined to be ≥1000 mg a.i./kg bw/day
Executive summary:

A study was conducted to determine the teratogenicity of the test substance, 'di-C16-18 and C18-unsatd. AAEMIM-MS' (active: 75%), according to EPA OPP 83 -3 Guideline, in compliance with GLP. During Gestation Day 6-15, four groups of 25 female Sprague CD rats/dose were dosed gavage at dose levels of 0, 100, 300, and 1000 mg/kg bw/day referring to 100% active substance, based on range finding study results conducted at doses up to 1875 mg/kg bw/day. Animals were sacrificed on Day 22. Examinations included daily observations of behaviour and clinical symptoms, daily food consumption, and body weight every three days. Upon sacrifice, number of corpora lutea, implantations and resorptions were examined. In the main study, no signs of maternal toxicity were observed, except one female in the 300 mg/kg bw/day group become moribund and was sacrificed on gestation Day 10. Also no foetal mortality was observed. Numbers of corpora lutea and resorptions were comparable. At 300 mg/kg bw/day and 1000 mg/kg bw/day dose groups 2 and 3 dams exhibited audible respiration during or subsequent to the treatment period. No treatment-related differences in gestational parameters including total number of implantations, number of viable and nonviable implants in any dose group. No treatment-related effects on gestational body weights and body weight gain, corrected body weight, corrected body weight gain, absolute and relative liver weight and gravid uterine weight were observed. Foetal body weights per litter were also not affected by treatment. Increased food consumption in the 100 mg/kg bw/day dose group was not considered to be related to treatment due to the lack of a dose-relationship. The animal in the 300 mg/kg bw/day dose group which was sacrificed due to moribund condition, had gas-filled intestines, mucoid fluid or material in the nose turbinates, abnormal material in the trachea and discoloured and consolidated lungs. One further animal in this dose group had no implants in one uterine horn. Two females in the 1000 mg/kg bw/day dose group contained blood in the uterus (detected with Hemastix reagent strips) another female had discoloured lungs (dark red) in all lobes. However, no statistically significant maternal toxic or embryotoxic/teratogenic effects were seen up to highest tested dose of 1000 mg/kg bw/day. Under the study conditions, the NOAEL for maternal and embryotoxic effects/teratogenicty in rats was established at 1000 mg/kg bw/day (Neeper-Bradley, 1992).