9-Octadecenoic acid (Z)-, reaction products with diethylenetriamine, di-Me sulfate-quaternized

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-05-13 to 1992-04-23

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

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

partially unsaturated IQAC, DMS quaternised

IUPAC Name:

partially unsaturated IQAC, DMS quaternised

Constituent 2

Reference substance name:

Imidazolium compounds, 2-(C15-17(odd numbered), C17-unsatd. alkyl)-1-[2-(C16-18(even numbered), C18-unsatd. amido)ethyl]-4,5-dihydro-N-methyl, Me sulfates)

IUPAC Name:

Imidazolium compounds, 2-(C15-17(odd numbered), C17-unsatd. alkyl)-1-[2-(C16-18(even numbered), C18-unsatd. amido)ethyl]-4,5-dihydro-N-methyl, Me sulfates)

Constituent 3

Reference substance name:

Fatty acids, C16-18 (even numbered) and C18 unsatd., reaction products with diethylene triamine, di-Me sulfate quaternized

EC Number:

937-237-2

Molecular formula:

Molecular formula cannot be given as substance is a mixture.

IUPAC Name:

Fatty acids, C16-18 (even numbered) and C18 unsatd., reaction products with diethylene triamine, di-Me sulfate quaternized

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Test animals
- Age: Males approx. 63 days; Females: approx. 56 days
- 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 hrs dark/12 hrs light; light period 5 – 17 hrs

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
ml / 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 days, from GD6 through GD15

Frequency of treatment:

once daily

Duration of test:

22 days (till gd21)

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

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

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 substance related due to their absence in the dose range finding study upto 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.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female became moribund and was sacrificed on GD10 in the 300 mg/kg bw/day dose group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No treatment-related effects on gestational body weights and body weight gain, corrected body weight and corrected body weight gain.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

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.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment-related effects on absolute and relative liver weight, and gravid uterine weight .

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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.

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.

Maternal developmental toxicity

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

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

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

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

Early or late resorptions:

no effects observed

Description (incidence and severity):

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)

Dead fetuses:

no effects observed

Description (incidence and severity):

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

Other effects:

no effects observed

Description (incidence and severity):

Fetal body weights per litter were not affected by treatment.

One animal in the 300 mg/kg bw/day dose group had no implants in one uterine horn.

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:

NOEL

Effect level:

>= 1 000 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Fetal body weights per litter were not affected by treatment.

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

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 bodyweights in litters at 100 mg/kg bw/day were not considered to be treatment-related due to the lack of a dose-response relationship.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Findings: Anterior arch of atlas poorly ossified; Statistical significance of affected litters in 1000 mg/kg bw/day dose group; % affected litters (71.4, 87.0, 86.4 96.0*).

Findings: Majority of forelimbs unossified; Statistical significance of affected litters in 300 mg/kg bw/day dose group; % affected litters (38.1, 26.1, 4.5**, 24.0).

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Findings: Excessive bleeding at umbilicus; Statistical significance of affected litters in 100 mg/kg bw/day dose group; % affected litters (23.8, 0*, 27.3, 40.0)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

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

Effect levels (fetuses)

Key result

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day

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 per dam)

-         Implantation sites (Total implants): 340 (15.5 per dam), 379 (16.5 per dam)*, 363 (15.7 per dam)**, 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 1000 mg/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 300 mg/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 in 100 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:

In conclusion the substance possessed no teratogenic properties, not even at the highest dose tested . Embryotoxicity was not observed.
Therefore the NOEL was 1000 mg active ingredient/kg bw/day. The NOEL for maternal toxicity was 1000 mg active ingredient/kg bw/day.

Executive summary:

In a developmental toxicity study according to OPP 83-3 the partially unsaturated IQAC, DMS quaternised (75%) was administered to 25 female Sprague CD rats/dose by gavage at dose levels of 0, 100, 300, and 1000 mg/kg bw/day referring to 100 % active substance from day 6 through 15 of gestation. The maternal NOEL is 1000 mg active substance/kg bw/day, based on the lack of effects indicating maternal toxicity. Likewise, the embryotoxic NOEL is 1000 mg active substance/kg bw/day, based on the lack of embryotoxic effects up to the highest dose level. The teratogenic NOEL is 1000 mg active substance/kg bw/day. The partially unsaturated IQAC, DMS quaternised showed no teratogenic properties (no incidence of malformations). The observed variations were isolated without dose relation or lower than in the control group and were therefore not regarded as treatment related. The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700) in rats. These findings are further backed by the lack of adverse effects in a dose range finding study which was conducted up to a dose level of 1875 mg/kg bw/day.