Poly(oxy-1,2-ethanediyl), α-[2-(dide- cylmethylammonio)ethyl]- .omega.- hydroxy-, propanoate (salt) (Bardap 26)

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not available

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

The test animals were male and female Sprague-Dawley rats (Crl:CD (SD) BR), obtained from Charles River Breeding Laboratories (MI, USA). The animals were acclimatised for approximately 2 weeks. Representative animals were selected for faecal examination, serum viral antibody analysis and histological examination of selected organs. The rats were housed in pairs during quarantine, and singly thereafter (except during cohabitation and lactation) in stainless steel mesh cages. From gestational day 20 through to weening, females were housed in plastic shoebox cages with bedding.
The rats were 6 weeks old at study initiation and weighed 204.2-205.8 g (males) and 153.3-155.2 g (females). Diet (Certified Ground Rodent Chow #5002) and tap water were provided ad libitum. Room temperature and humidity were monitored, and a 12 hour light/dark cycle was maintained.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

The test substance was mixed direcly into the animal diet (fed ad libitum). All weights of the test material were corrected for percent active ingredient for diet preparation, and loss of ethanol during diet preparation was also taken into account. The test diets were stored at room temperature.

Details on mating procedure:

Rats were mated one male to one female for 3 weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration of the test substance in the diet, homogeneity and stability were determined using gas chromatography with Nitrogen-Phosphorous detection. Homogeneity was confirmed, and the test diets were demonstrated to be stable for 21 days when stored in closed polyethylene containers and for at least 14 days when stored in open glass jars at room temperature. Periodic analyses of the test diets indicated that the mean test substance concentrations in the diet for the 300, 750 and 1500 ppm levels were 96.2-108.8%, 98.8-108.8% and 94.5-109.2% of nominal, respectively.

Duration of treatment / exposure:

F0: 27 weeks (from 1st prebreed dose to last F0 sacrifice)
F1: 32 weeks (from 1st F1B wean to last F1B sacrifice)
F2B: to weaning

Frequency of treatment:

Continuous - ad libitum dietary exposure

Details on study schedule:

After a 10-week pre-breed period, rats were mated (one male to one female) for three weeks to produce the F1A generation. The observation of a dropped copulation plug or the presence of vaginal sperm was considered evidence of successful mating. Exposures continued through mating, gestation, parturition and lactation. At least 10 days after the weaning of the F1A litters, the F0 parents were paired again to produce the F1B generation. At weaning, 28F1B weanlings/sex/group were selected to produce the F2 generation, and were then exposed to the same dietary concentrations of Didecyldimethylammonium Chloride as their parents for 10 weeks. After their pre-breed exposure, F1 animals were paired as described above to produce F2A and F2B litters.

Dose / conc.:

300 ppm

Remarks:

Basis:
nominal in diet

Dose / conc.:

750 ppm

Remarks:

Basis:
nominal in diet

Dose / conc.:

1 500 ppm

Remarks:

Basis:
nominal in diet

No. of animals per sex per dose:

28/sex/group

Control animals:

yes, plain diet

Details on study design:

Initial animals (F0) were randomly assigned to groups using a computer generated stratified body weight programme. Only healthy animals with a body weight ±20% of the mean weight for each sex were included in the study.

Positive control:

Not applicable.

Parental animals: Observations and examinations:

Detailed clinical observations were performed once each week. Observations for overt clinical signs were made twice daily on days when detailed observations were not conducted. Observations for mortality were made twice daily.
Bodyweights were recorded weekly during the pre-beeding period and during mating for both sexes. Bodyweights were recorded for females on gestational day (gd) 0, 6, 15 and 20 and on postnatal day (pnd) 0, 7, 14 and 21.
Food consumption was recorded weekly during the pre-breeding period for both sexes, and for females in 3 or 4 day intervals throughout gestation and to pnd 4.

Oestrous cyclicity (parental animals):

Not determined.

Sperm parameters (parental animals):

Not determined.

Litter observations:

All pups were weighed on postnatal day (pnd) 1, 4, 7 and 14, and at weaning (day 21).

Postmortem examinations (parental animals):

All F0 and F1 parental animals were necropsied and examined for gross lesions. Reproductive tissues and other tissues with gross lesions identified as potentially related to treatment were harvested. All these tissues from the high dose and control groups and any tissues or organs showing gross alterations from the low and mid dose groups were examined histologically. A complete gross necropsy and histopathologic examination were conducted for any parental animals dying on test.

Postmortem examinations (offspring):

Examination for gross lesions was performed on any pup appearing abnormal or dying on test. Ten F1A, F1B and F2 weanlings/sex/group were randomly selected and examined for gross lesions. Remaining nonselected F1 and F2 pups at weaning were euthanised and discarded after the necropsy of the selected pups.

Statistics:

The results of the quantitative continuous variables (e.g., body weights, food consumption, etc.) were intercompared for the three treatment groups and one control group by use of Leaven’s test for equal variances, analysis of variance and (pooled or separate variance) t-test. Non-parametric data were statistically evaluated using the Kruskal-Wallis test followed by the Mann-Whitney U test for pairwise comparisons when appropriate. Frequency data were compared using the Fisher’s exact test.

Reproductive indices:

Mating index, fertility index, gestational index, live birth index, 4-day survival index, 7-day survival index, 14-day survival index, 21-day survival index, lactation index, 28-day survival index.

Offspring viability indices:

See above.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced at 1500 ppm a.s.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

reduced at 1500 ppm a.s.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No significant signs of toxicity during the pre-breed, mating, gestation or lactation periods at any dose for either generation. There were no mortalities.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
F0 and F1 10-Week Pre-mating Exposure: No treatment-related effects on body weights were observed at 300 or 750 ppm a.s.. Males and females at 1500 ppm a.s. had reductions in body weight beginning one week after treatment. Body weight gain was also reduced.
F0 and F1 Gestation/Lactation: No treatment-related effects on body weights were observed at 300 or 750 ppm a.s.. Females in the 1500 ppm a.s. group showed significant reductions in body weights but no body weight gain reductions during the first (producing the F1A and F2A litters) breeding period. Body weight but not weight gain was also reduced throughout lactation. During the second breeding (producing the F1B and F2B litters), gestational body weights and weight gains appeared to be lower than control and lactational body weights but not weight gains were reduced.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
F0 and F1 10-Week Pre-mating Exposure: No treatment-related effects on food consumption were observed at 300 or 750 ppm a.s.. Food consumption in males and females was reduced throughout the pre-breed periods for the 1500 ppm a.s. group.
F0 and F1 Gestation/Lactation: Food consumption during gestation and lactation for both breeding periods was unaffected by test substance treatment at any dose.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Reproductive parameters were unaffected by treatment for all groups during the first and second breeding of both the F0 and F1 animals.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No treatment-related findings were observed.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment-related findings were observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related findings were observed.

OTHER FINDINGS (PARENTAL ANIMALS)
No other findings were reported.

Key result

Dose descriptor:

NOAEL

Effect level:

750 ppm (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Equivalent to ca. 928 ppm test substance. Based on reduced body weights and food consumption at 1500 ppm a.s.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced at 1500 ppm a.s.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

CLINICAL SIGNS (OFFSPRING)
There were no signs of toxicity in the F1A, F1B, F2A or F2B animals.

BODY WEIGHT (OFFSPRING)
No treatment-related effects on body weights were observed at 300 or 750 ppm. F1A litters exhibited reduced body weights and weight gains from pnd 14 through 28 at 1500 ppm. A similar effect was observed for F1B, F2A and F2B pups.

ORGAN WEIGHTS (OFFSPRING)
There were no treatment-related findings in the F1A, F1B, F2A or F2B weanling animals at necropsy.

GROSS PATHOLOGY (OFFSPRING)
There were no treatment-related findings in the F1A, F1B, F2A or F2B weanling animals at necropsy.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

750 ppm (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Equivalent to ca. 928 ppm test substance. Based on reduced body weights and food consumption at 1500 ppm a.s.

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

750 ppm (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Equivalent to ca. 928 ppm test substance. Based on reduced body weights at 1500 ppm a.s.

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

750 ppm

Treatment related:

yes

Conclusions:

These results indicate that continuous exposure to test item in the diet for two generations in Sprague-Dawley (CD®) rats resulted in no adverse reproductive effects. Parental toxicity was observed at 1500 ppm, limited to body weight reduction, weight gain depression and decreased food consumption. Postnatal toxicity was observed at 1500 ppm, consisted of reduced pup body weights. The adult effect levels are equivalent to 112.6 mg of test chemical per kg body weight at 1500 ppm. The "no observable effect level" (NOEL) for adults in this study was 750 ppm. The NOEL for offspring in this study was 750 ppm (males: 38 - 79 mg/kg bw/day and females: 49 - 82 mg/kg bw/day), indicating no increased risk to offspring in the absence of maternal effects.

Executive summary:

The study was carried out in accordance with EPA OPP 83-4. Sprague-Dawley rats were given diets containing Bardac 2280 ( Didecyldimethylammonium Chloride in aqueous/alcohol solution) at concentrations of 0, 300, 750 and 1500 ppm a.s.. A 10-week pre-breed exposure was used for both the F0 and F1 generations. Two litters per generation were produced. Body weights were decreased in males and females at 1500 ppm for most of the pre-breeding exposure period as well as for the F1A, F1B, F2A, and F2B offspring during lactation. Food consumption was also reduced during the pre-breeding periods for both the F0 and F1 parental animals. No other treatment-related effects were observed including on any reproductive parameters. On the basis of these results the NOAEL (parental, F1 offspring and F2 offspring) was considered to be 750 ppm a.s. (equivalent to ca. 928 ppm test substance). It was concluded that the test substance was not toxic to reproduction under the conditions of the study.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

112 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

A high-quality rat multigeneration study is available for the read-across substance DDAC.No effects on reproduction were seen at the highest dose level of 1500 ppm (112 mg/kg bw/d)

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Adequate studies evaluating teratogenicity have been conducted on the chemical and structural analogue, Didecyldimethylammonium Chloride. In view of the chemical and structural similarities, it is considered that the available data are adequate for N,N-Didecyl-N-methyl-poly(oxyethyl)ammonium Propionate.

In a two-generation dietary feeding study with rats (Neeper-Bradley, 1991), parental toxicity was observed at the highest dose (1500 ppm a.s.; ~112 mg a.s./kg bw/day) and was limited to weight gain depression and reduced feed consumption. Pup weights were also reduced at this dosage. There were no effects on reproduction in this study and toxicity to the offspring (reduced body weight) occurred only in the presence of maternal toxicity. On the basis of these results the NOAEL (parental, F1 offspring and F2 offspring) was considered to be 750 ppm a.s. (equivalent to ca. 928 mg test substance/kg bw).

Short description of key information:
A high-quality rat multigeneration study is available for the read-across substance DDAC.No effects on reproduction were seen at the highest dose level of 1500 ppm (112 mg/kg bw/d)

Justification for selection of Effect on fertility via oral route:
A single study is available for this endpoint.

Effects on developmental toxicity

Description of key information

In a developmental toxicity study with rats the NOAEL for maternal toxicity was 1 mg a.s./kg bw/day and for developmental toxicity was at least 20 mg a.s./kg bw/day. In a developmental toxicity study with

rabbits the NOAELs were 1.0 mg a.s/kg bw/day for maternal toxicity and 3 mg a.s./kg bw/day for developmental toxicity.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

May-July 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

The test animals were female New Zealand White Rabbits obtained from Hazelton-Dutchland Laboratories, USA. They were quarantined for approximately 2 weeks. Representative animals were selected for faecal analysis. The rabbits were single housed in stainless steel wire mesh cages with food (Agway Prolab Certified Chow) and municipal water provided ad libitum. Males from the test facility breeding colony (originally from the same supplier) were used for mating. Temperature was maintained at 66-72°F and relative humidity at 40-60%, with a 12 hour light/dark cycle. They were approximately 6 months old at study initiation and weighed at least 2.5 kg.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Millipore

Details on exposure:

The test substance was administered orally by gavage in Millipore water. The concentration of the test substance in the vehicle was 0, 0.5, 1.5 and 5 mg/ml. The dosing formulations were based on active ingredient content, and were prepared once during the study. The dosing volume was 2.0 ml/kg bw.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each test solution was analysed for test substance content using gas chromatography with nitrogen-phosphorous detection. The formulations were shown to homogenous and stable for at least 21 days. Test substance concentrations were 94.0-101.4% of target concentrations.

Details on mating procedure:

Females were mated 1:1 with proven fertile males. The day mating was observed was designated GD 0 and females were housed singly again from this point.

Duration of treatment / exposure:

Gestation days 6-18

Frequency of treatment:

Daily during the exposure period

Duration of test:

Until Day 29 of gestation

No. of animals per sex per dose:

16 dams/groups

Control animals:

yes, concurrent vehicle

Details on study design:

Sixteen mated females were assigned to each experimental group by a computer-generated randomisation procedure stratified by body weight on GD0, so that groups were equivalent in mean body weight and body weight range.

Maternal examinations:

The dams were observed twice daily for clinical signs and mortality. Bodyweights were recorded on gestation days 0, 6, 13, 19, 24 and 29. Gross necropsy was performed at study termination (GD 29). The gravid uterus, ovaries (including corpora lutea), cervix, vagina, and abdominal and thoracic organs and cavities were examined grossly. The lumen and lining of the oesophagus, stomach and trachea were examined for any indications of irritation. Organ weights were recorded for the liver and uterus.

Ovaries and uterine content:

Ovarian corpora lutea were counted. The uterus was examined externally for signs of haemorrhage. All live and dead foetuses and resorption sites were recorded. Uteri from females that appeared non gravid were placed in 10% ammonium sulphide solution for confirmation of pregnancy status and for detection of early resorptions.

Fetal examinations:

All live foetuses were immediately euthanised then weighed, sexed and examined for external malformations. All foetuses were examined for skeletal abnormalities using alizarin red S, and visceral abnormalities. Half the foetuses were used for examination of craniofacial structures following fixing in Bouin's solution.

Statistics:

Levene’s test for equal variances, analysis of variance, and t-tests with Bonferroni probabilities for pairwise comparisons. Nonparametric data were analyzed with Kruskal-Wallis test followed by Mann-Whitney U test when appropriate. Incidence data were compared using Fisher’s Exact Test.

Indices:

No information available.

Historical control data:

No information available.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Four of 16 does died at 10 mg a.s./kg bw/d prior to gestation day 13; all were pregnant. One doe at 10 mg.kg bw/d and two at 1.0 mg/kg bw/d delivered early and were removed from the study. No does aborted. Treatment-related clinical signs were observed at 3.0 and 10 mg a.s./kg bw/d, primarily related to audible breathing and hypoactivity. Statistically significant reduced body weight gain was observed at the high dose during gestation days 6-13, reduced weight gain was also observed for gestation days 13-19. At 3.0 mg a.s./kg bw/d, maternal weight gain was significantly reduced for gestation days 13-19 and 6-19. There were no abnormalities detected at scheduled necropsy. Gross necropsy of the does that died revealed sloughing of the oesophageal lining, sloughing, haemorrhage and distention of the glandular portion of the stomach, sloughing and haemorrhage of the non-glandular portion of the stomach, mucoid material in the trachea, colour change, firm texture and/or consolidation of the lungs, gas filled intestines, reticular pattern in the liver and distended urinary bladder.

Key result

Dose descriptor:

NOAEL

Effect level:

1 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

maternal abnormalities

Key result

Dose descriptor:

NOAEL

Effect level:

3 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Pregnancy rate was approximately equivalent across all groups. Reduced foetal weights were observed at 10 mg a.s./kg bw/d. No malformations were noted at gross necropsy. There were no treatment-related skeletal or visceral variations or malformations. The incidence of dead foetuses per litter was significantly increased at 10 mg/kg bw/d.

Abnormalities:

not specified

Key result

Developmental effects observed:

no

No developmental toxicity, including teratogenicity, was observed at any dosage employed.

Conclusions:

The test substance was not teratogenic. The NOAEL for maternal toxicity was 1 mg a.s./kg bw/d (equivalent to ca. 1.2 mg test substance/kg bw/d); the NOEL for developmental toxicity was 3 mg a.s./kg bw/d (equivalent to ca. 3.7 mg test substance/kg bw/d).

Executive summary:

The teratogenic potential of Bardac 2280 was evaluated in accordance with EPA OPP 83-3. Pregnant rabbits were treated with the test substance by gavage, at doses of 0, 1, 3 and 10 mg a.s./kg bw/d on gestation days 6 -18. The does were sacrificed at gestation day 29 and the foetuses were examined for visceral and skeletal variations and malformations.

Treatment-related clinical signs were observed at 3 and 10 mg a.s./kg bw/d primarily related to audible breathing and hypoactivity. Four of 16 does died at 10 mg a.s./kg bw/d prior to gestation day 13. Reduced body weight gain was observed at the mid and high dose groups. An increased incidence of dead foetuses per litter and reduced foetal body weights per litter was observed at 10 mg/kg bw/d, but these effects were considered to be associated with the maternal toxicity observed at this dose.

It was concluded that the test substance was not teratogenic. The NOAEL for maternal toxicity was 1 mg a.s./kg bw/d (equivalent to ca. 1.2 mg test substance/kg bw/d); the NOEL for developmental toxicity was 3 mg a.s./kg bw/d (equivalent to ca. 3.7 mg test substance/kg bw/d).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

3 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

High quality, guideline- and GLP-compliant studies are available for the read-across substance DDAC in the rat and rabbit. No developmental toxicity was observed in either species.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Adequate studies evaluating teratogenicity have been conducted on the chemical and structural analogue, Didecyldimethylammonium Chloride. In view of the chemical and structural similarities, it is considered that the available data are adequate for N,N-Didecyl-N-methyl-poly(oxyethyl)ammonium Propionate.

In a developmental toxicity study with rats (Neeper-Bradley, 1991), maternal toxicity, similar to that seen in other toxicity studies with rats, was observed at 10 or 20 mg a.s./kg bw/day by gavage. The NOAEL for maternal toxicity was 1 mg a.s./kg bw/day and for developmental toxicity was at least 20 mg a.s./kg bw/day. 

In a developmental toxicity study with rabbits (Tyl, 1989), 25% of the does died at the highest dose (10 mg a.s./kg bw/day) exhibiting signs consistent with severe gastrointestinal effects. Maternal toxicity at 3 mg a.s./kg bw/day included reduced weight gain. Foetal mortality and reduced foetal body weight were observed at the high dose only. The NOAELs were 1.0 mg a.s./kg bw/day for maternal toxicity and 3 mg a.s./kg bw/day for developmental toxicity.

No teratogenicity was induced by Bardac 2280 in either species. Therefore, it is concluded that Bardap 26 will not induce teratogenicity.

Justification for selection of Effect on developmental toxicity: via oral route:
High quality, guideline- and GLP-compliant studies are available for the read-across substance DDAC in the rat and rabbit. Although the rat is the preferred species, a lower NOAEL was observed in the rabbit study.

Justification for classification or non-classification

Didecyldimethylammonium Chloride, a structural analogue of N,N-Didecyl-N-methyl-poly(oxyethyl)ammonium Propionate, was not teratogenic in rats and rabbits and was not toxic to reproduction in rats. Therefore it is concluded that N,N-Didecyl-N-methyl-poly(oxyethyl)ammonium Propionate does not require classification for reproductive or developmental toxicity.