Fatty acids, C16-18 (even numbered), reaction products with tetraethylenepentamine, acetates (salts)

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:

68122-86-1 (partially unsaturated IQAC, DMS quaternised)

IUPAC Name:

68122-86-1 (partially unsaturated IQAC, DMS quaternised)

Constituent 2

Reference substance name:

partially unsaturated IQAC, DMS quaternised

IUPAC Name:

partially unsaturated IQAC, DMS quaternised

Constituent 3

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)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Test animals
- Source: Charles River Breeding Laboratories, Inc. (Partage, MT)
- 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 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)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

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 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.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

300 mg/kg bw/day dose group:

One female became moribund and was sacrificed on gd10.

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, 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.

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. 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.

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

Details on maternal toxic effects:

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)

* 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

Effect levels (maternal animals)

Key result

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

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.

Reduction in number of live offspring:

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)

External malformations:

no effects observed

Description (incidence and severity):

Malformations: none significantly different from Control

External variations: none significantly different from control

Skeletal malformations:

no effects observed

Description (incidence and severity):

Malformations: none significantly different from Control

Skeletal variations:
- 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*)
- 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)

* 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

Visceral malformations:

no effects observed

Description (incidence and severity):

Malformations: none significantly different from Control

Soft Tissue variations:
- 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)

Effect levels (fetuses)

Key result

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

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

Summary of distribution and fate

group: mg/kg bw/d		0	100	300	1000
Females on study		25	25	25	25
Mortality	Total %	0 0.0	0 0.0	1 * 4.0	0 0.0
Females that âborted	Total %	0 0.0	0 0.0	0 0.0	0 0.0
Females that delivered	Total %	0 0.0	0 0.0	0 0.0	0 0.0
Femaled removed from study	Total %	0 0.0	0 0.0	0 0.0	0 0.0
Females examined at lapratomy	Total %	25 100.0	25 100.0	24 96.0	25 100.0
Nonpregnant	Total %	3 12.0	2 8.0	1 4.2	0 0.0
Pregnant	Total %	22 88.0	23 92.0	23 95.0	25 100.0
Females with non-viable implants only	Total %	1 4.5	0 0.0	1 4.3	0 0.0
Females with viable fetuses	Total %	21 95.5	23 100.0	22 95.7	25 100.0

* Female sacrificed moribund on GD10

 

Summary of gestational parameters

group: mg/kg bw/d		0	100	300	1000
Corpora lutea	Mean SD N	16.1 2.02 21	16.5 3.57 23	15.7 3.53 23	15.5 2.50 25
Total implants	Mean SD N	15.5 3.81 22	16.5 3.80 23	15.7 3.56 23	15.3 2.69 25
%implantation loss a	Mean SD N	0.3 1.42 22	1.9 4.65 23	3.1 10.52 23	2.3 4.56 25
Viable implants	Mean SD N	14.9 4.0 22	15.5 3.50 23	14.8 3.68 23	14.7 2.58 25
Non-viable implants	Mean SD N	0.6 0.73 22	1.0 1.17 23	0.9 0.92 23	0.6 0.71 25
Early resorptions	Mean SD N	0.6 0.73 22	0.9 1.04 23	0.7 0.92 23	.0.6 0.72 25
Late resorptions	Mean SD N	0.0 0.0 22	0.0 0.21 23	0.1 0.42 23	0.0 0.0 25
Dead fetuses	Mean SD N	0.0 0.0 22	0.0 0.21 23	0.0 0.42 23	0.0 0.0 25
%live fetuses	Mean SD N	92.0 21.10 22	94.5 6.29 23	90.6 20.54 23	96.2 4.63 25
Sex ratio (%male fetuses)	Mean SD N	48.7 11.94 22	47.4 17.52 23	48.2 16.25 22	45.4 14.13 25
Fetal body weights per litter [g]
All fetuses	Mean SD N	5.564 0.3494 21	5.366 0.2623 23	5.516 0.3768 22	5.597 0.2476 25
Male fetuses	Mean SD N	5.697 0.3746 21	5.499 * 0.2400 23	5.685 0.3500 22	5.750 0.2448 25
Female fetuses	Mean SD N	5.439 0.3360 21	5.230 * 0.2224 22 b	5.376 0.3980 22	5.479 0.2703 25

* significantly different from control group (p</0 0.05)

a percent preimplantation loss = [(corpora lutea - total implants)/corpora, lutea] X 100,

b The N is reduced because one litter consisted of male fetuses only.

Applicant's summary and conclusion

Conclusions:

In conclusion the test item partially unsaturated IQAC, DMS quaternised 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 prenatal developmental toxicity study according to EPA OPP 83-3 timed-pregnant CD® (Sprague-Dawley) rats were administered the test item partially unsaturated IQAC, DMS quaternised (75%) by gavage on gestation days (gd) 6 through 15. Twenty-five copulation plug-positive females per group were dosed with undiluted test item at dose levels corresponding to 100, 300, and 1000 mg/kg/day of active ingredient. These dose levels were selected on the basis of results from a dose range-finding study in which no maternal toxicity or evidence of teratogenicity was observed at dose levels as high as 1875 mg/kg/day. An additional 25 females, assigned to the control group, received Milli-Q® water at a dose volume equivalent to that used in the high dose group. Clinical observations were made daily (twice daily during dosing), and maternal body weights were measured on gd 0, 6, 9, 12, 15, 18 and 21. Maternal food consumption was measured at 3-day intervals throughout gestation; gd 0-21. At scheduled sacrifice on gd 21, the dams were evaluated for liver and gravid uterine weights, number of corpora lutea and’ number and status of implantation sites (including early and late resorptions, dead fetuses and live fetuses).

All live and dead fetuses were dissected from the uterus, weighed and examined externally for malformations and variations and gender determinations.

Approximately one-half of the live fetuses in each litter were examined for visceral and craniofacial malformations and variations. The remaining one half of the fetuses were stained with alizarin red S and were examined for skeletal malformations and variations.

The pregnancy rate was equivalent across groups and ranged from 88-100%. No females aborted or delivered early. At scheduled sacrifice, 3 females in the control group; 2 females in the 100 mg/kg/day group and 1 female in the 300 mg/kg/day group were found to be nonpregnant. One female from the control group and 1 female from the 300 mg/kg/day group contained no viable fetuses at scheduled sacrifice. 21 to 25 live litters were available for evaluation from each group.

One female in the 300 mg/kg/day treatment group became moribund and was sacrificed on gd 10. Two to 3 dams in the 300 and 1000 mg/kg/day treatment groups exhibited audible respiration during or subsequent to the treatment period. None of these observations were considered to be test substance related. There were no treatment-related effects on food consumption, gestational body weight and body weight gain, corrected body weight, corrected body weight gain, and gravid uterine weight. No treatment-related differences in gestational parameters including total number of implantations, number of viable and nonviable implants, were observed in any dose group, fetal body weights per litter were not affected by treatment. No treatment-related malformations or variations were observed in this study.

Administration of the test item by gavage to pregnant Charles River CD® rats during organogenesis resulted in no treatment-related maternal toxicity, embryotoxicity, teratogenicity, or developmental delay. In this study, the ‘no observable effect level" (NOEL) for maternal toxicity as well as for developmental toxicity was at least 1000 mg/kg/day.

 

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.