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

Endpoint summary

Description of key information

The 90-day oral (diet) NOAEL of Bardap 26 in rats was 391 mg/kg bw/day 
The 90-day dermal NOAEL of Didecyldimethylammonium Chloride in rats was 14.9 mg/kg bw/d).
The 8-week oral (gavage) NOAEL of Didecyldimethylammonium Chloride in dogs was 30 mg/kg bw/d
The 52-week oral (gavage) NOAEL of Didecyldimethylammonium Chloride in dogs was 10 mg a.s./kg bw/d (equivalent to 12.4 mg test substance/kg bw/d).
The 104-week oral (diet) NOAEL of Didecyldimethylammonium Chloride in rats was 32 and 41 mg/kg bw/d for males and females, respectively.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 1998 to March 1999

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

yes

Remarks:

specific neurotoxicity functional observations are not included

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (Testing Guidelines for Toxicology Studies 59 No 4200)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

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 Crl:CD BR rats obtained from Charles River (UK) Limited, Kent, UK. They were 5-8 weeks old and weighed 170-217 g (males); 149-189 g (females). The animals were acclimatised for 11 days. They were housed in groups of up to four by sex in polypropylene grid-floor suspended cages. A ground diet (Rat and Mouse SQC Ground Diet No. 1, SDS Ltd., Essex UK) was provided ad libitum, except during urine collection when food was withdrawn overnight. Mains water was supplied ad libitum. The animal room was maintained to provide a temperature of 21±2°C, relative humidity of 55±15%, at least 15 air changes per hour, and lighting was provided on a 12 hour light/dark cycle.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The test substance was administered in the diet for 90 days. The test susbtance was mixed directly into the diet. Diet formulations were prepared prior to treatment then twice during the three month period. The mixed diet was stored in double black plastic bags in covered plastic bins.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity of each diet formulations was determined. Test substance concentrations in the diet admixtures were determined using GCMS. The results indicated that the admixtures were stable, homogenous, and the mean prepared diet concentrations were acceptable for the purposesof the study.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Continuous - dietary exposure

Dose / conc.:

42 mg/kg bw/day (nominal)

Dose / conc.:

127 mg/kg bw/day (nominal)

Dose / conc.:

391 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/sex/group

Control animals:

yes, plain diet

Details on study design:

The animals were randomly allocated to treatment groups using random letter tables and the group mean body weights were then determined to ensure similarity between treatment groups.

Animals were exposed to the test substance in the diet for 90 days. Survivors were sacrificed at study termination (Day 90).

Positive control:

Not applicable.

Observations and examinations performed and frequency:

The rats were observed for clinical signs and mortality once daily. Individual bodyweights were recorded on Day 0 and weekly thereafter. Food consumption was determined for each cage weekly. Water consumption was assessed daily by visual inspection. ophthalmoscopic examinations were conducted for all rats at the start of treatment, and for control and high dose rats at Week 12.
Blood was collected at study termination for haematology and clinical chemistry determinations. The following haematology parameters were determined: haemoglobin, erythrocyte count, haematocrit, MCH, MCV, MCHC, total leukocyte count, differential leukocyte count, platelet count and reticulocyte count. Prothrombin time and activated partial thromboplastin time were assessed. The following clinical chemistry parameters were determined: urea, glucose, total protein, globulin, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium., magnesium, inorganic phosphorous, gamma glutamyltransferase, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, sorbitol dehydroferase, creatinine, total cholesterol, total bilirubin.
Urine was collected from all animals at Week 13 for urinalysis. The following parameters were determined: volume, bilirubin, specific gravity, pH, urobilinogen, reducing substances, protein, blood, glucose, ketones, microscopic examination of sediment.

Sacrifice and pathology:

All surviving animals were sacrificed at study termination and subject to gross necropsy. The following organs from terminal kill animals were weighed: adrenals, brain, epididymides, heart, kidneys, liver, lungs, ovaries, spleen, testes, thymus and uterus. The following tissues were processed for histological examination: adrenals, aorta, bone and bone marrow (femur and sternum), brain, caecum, colon, duodenum, epididymides, eyes, gross lesions and masses, heart, ileum, jejunum, kidneys, larynx, liver, lungs, lymph nodes, mammary gland, muscle, nasal cavity, oesophagus, ovaries, pancreas, pharynx, pituitary, prostate, rectum, salivary glands, sciatic nerve, seminal vesicles, skin, spinal cord, spleen, stomach, testes, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder, uterus. Histopathology was carried out for all tissues from control and high dose rats, and for the lungs, liver and kidneys from all rats at 500 and 1500 ppm (352 and 1056 ppm a.s.).

Other examinations:

No other examinations performed.

Statistics:

ANOVA, Dunnett’s test, Levene’s test, Kruskal Wallis ANOVA and Mann Whitney U test.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinically observable signs of toxicity were detected.

Mortality:

no mortality observed

Description (incidence):

One control male died during blood sampling procedures conducted on Day 90 of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 4500 ppm showed a reduced bodyweight gain over the first six weeks of treatment when compared with controls with the exception of Week 3 for females. Animals of either sex treated with 1500 or 500 ppm showed a similar bodyweight development to controls throughout the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

reduced at 4500 ppm

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 4500 ppm showed a reduced dietary intake throughout the study period when compared with controls with the exception of Week 8 for females. Animals of either sex treated with 1500 or 500 ppm showed a similar dietary intake to controls throughout the study.

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

no effects observed

Description (incidence and severity):

No overt intergroup differences were detected.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related ocular changes were detected.

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related haematological changes were detected.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Reduced plasma total protein and globulin, and increased plasma alanine aminotransferase and aspartate aminotransferase were observed at 3168 ppm a.s. (both sexes). Reduced plasma cholesterol was observed in 4500 ppm females.
Animals of either sex treated with 1500 or 500 ppm showed no such changes in the blood chemical parameters measured.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were detected.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Reduced absolute liver weights were observed at 3168 ppm a.s. (both sexes). Reduced relative liver weights were observed in 4500 ppm males.
No treatment-related organ weight changes were detected among animals of either sex treated with 1500 or 500 ppm.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Three 4500 ppm females had a small spleen. No treatment-related macroscopic abnormalities were detected among animals of either sex treated with 1500 or 500 ppm.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related microscopic abnormalities were detected.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no treatment-related mortalities, however one control male died during blood sampling on Day 90. No clinical signs of toxicity were observed. Isolated occurrences of hair loss and/or red/brown staining of the external body surface were observed among animals of either sex from all groups; these signs occasionally occur in group housed rats and were therefore not considered to be of toxicological significance.

BODY WEIGHT AND WEIGHT GAIN
Body weight gain was reduced in both sexes of animals treated with 4500 ppm during the first 6 weeks of treatment (except for females in Week 3). Body weight development returned to normal in these animals after week 6, although these animals completed the treatment period with a lower terminal body weight than the controls. Animals in the 500 and 1500 ppm groups showed similar body weight gains to the controls.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Reduced food intake was observed at 4500 ppm (both sexes) throughout treatment, except in females at Week 8.

FOOD EFFICIENCY
Food efficiency was reduced in 4500 ppm females during the first week of treatment.

WATER CONSUMPTION
There were no treatment-related changes.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related changes.

HAEMATOLOGY
There were no treatment-related changes. Females treated with 4500 ppm showed a slight (but significant) reduction in total leukocyte count and a significant increase in haematocrit compared to controls. However there were no concomitant changes in associated haematological parameters or any histopathological correlates, therefore the slight difference observed was not considered to be toxicologically relevant.

CLINICAL CHEMISTRY
Reduced plasma total protein and globulin, and increased plasma alanine aminotransferase and aspartate aminotransferase were observed at 4500 ppm (both sexes). Reduced plasma cholesterol was observed in 4500 ppm females. Reduced plasma sorbitol dehydrogenase was observed in the 4500 ppm males, but was considered unlikely to be of toxicological significance.

URINALYSIS
There were no treatment-related changes.

ORGAN WEIGHTS
Reduced absolute liver weights were observed at 4500 ppm (both sexes). Reduced relative (to terminal body weight) liver weights were observed in 4500 ppm males. It was considered likely that these changes were primarily associated with the reduced terminal body weight observed in this group, particularly in the absence of any histopathological correlates, but since a slght effect on relative liver weight was observed, a relationship to treatment could not be discounted. Other incidental differences were noted in the 4500 ppm group, however these findings were considered to be related to the effects on body weight, and there were no histopathological correlates - therefore these findings were considered to be of no toxicological significance.

GROSS PATHOLOGY
Three 4500 ppm females had a small spleen. Two 4500 ppm males and one 1500 ppm male had a malformed spleen, but this was considered to be a congenital abnormality often seen in laboratory rats and was therefore not considered to be relevant.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment-related changes.

Key result

Dose descriptor:

NOAEL

Effect level:

391 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOEL

Effect level:

127 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Key result

Critical effects observed:

not specified

Table 1. Group mean body weight change (g)

Study Week	Dose level (ppm)
	0		500		1500		4500
	Male	Female	Male	Female	Male	Female	Male	Female
1	58	28	59	25	63	26	42***	13***
2	52	21	49	21	54	19	43	15*
4	36	16	35	16	34	15	27***	11
6	23	8	25	10	22	7	18	5
0-13	290	104	303	114	294	110	225	64

    * p < 0.05
*** p < 0.001

 

Table 2. Group mean food consumption (g/rat/day)

Study Day	Dose level (ppm)
	0		500		1500		4500
	Male	Female	Male	Female	Male	Female	Male	Female
7	216	170	206 (-5)	176 (+4)	211 (-2)	157 (-8)	157 (-27)	117 (-31)
28	223	166	218 (-2)	170 (-2)	223 (0)	169 (+2)	189 (-15)	137 (-17)
56	213	157	205 (-4)	175 (+11)	211 (-1)	162 (+3)	175 (-18)	147 (-6)
90	145	95	155 (+7)	100 (+5)	124 (-14)	104 (+9)	113 (-22)	76 (-20)

% of control in parenthesis

Table 3. Group mean plasma clinical chemistry

Parameter	Dose level (ppm)
	0		500		1500		4500
	Male	Female	Male	Female	Male	Female	Male	Female
Total protein (mg/dl)	7.02	7.28	7.03	7.24	6.95	7.18	6.72*	6.88*
Total globulin (g/dl)	3.91	3.76	3.91	3.75	3.79	3.73	3.64*	3.54**
Alanine aminotransferase (IU/l)	44	43	39	33	43	32	82***	59**
Aspartate aminotransferase (IU/l)	89	102	89	86	85	88	107***	111
Chloesterol (mg/dl)	70	78	61	82	56	73	63	62*

    *p < 0.05
 ** p < 0.01
*** p < 0.001

 

Table 4. Group mean liver weight (g) and body weight relative liver weights (%)

Weight	Dose level (ppm)
	0		500		1500		4500
Liver: absolute	15.6175	8.9095	15.9903	8.9487	16.3245	8.6200	11.1183***	7.5950**
Body weight relative	3.0598	3.0852	3.1008	2.9993	3.1752	2.9401	2.5798**	3.1173

  ** p < 0.01
*** p < 0.001

Conclusions:

There was no purity correction incorporated for this study, the actual test material concentration was calculated at those dose levels.
NOEL = 127 mg/kg/day (1500 ppm)
NOAEL = 391 mg/kg/day (4500 ppm)

Executive summary:

Groups of Sprague Dawley rats (10/sex/dose level) were fed Bardap 26 (N,N-Didecyl-N-methyl-poly(oxyethyl)ammonium Propionate aqueous/alcohol solution) for 90-days at 0, 500, 1500 or 4500 ppm according to OECD 408.

There were no treatment-related mortalities or clinical signs of toxicity in any animal during the study. There were no treatment-related effects on water consumption (visual inspection), haematology or urinalysis and there were no treatment-related changes detected during ophthalmoscopic examination. Body weight gain was reduced in both sexes during the first 6 weeks of treatment (except for females in Week 3). Reduced food intake was observed at 4500 ppm (both sexes) throughout treatment, except in females at Week 8. Reduced plasma total protein and globulin, and increased plasma alanine aminotransferase and aspartate aminotransferase were observed at 4500 ppm (both sexes). Reduced plasma cholesterol was observed in 4500 ppm females. Reduced absolute liver weights were observed at 4500 ppm (both sexes). Reduced relative liver weights were observed in 4500 ppm males. Gross necropsy revealed three 4500 ppm females had a small spleen.

There was no purity correction incorporated for this study, the actual test material concentration was calculated at those dose levels.

The NOEL = 1500 ppm (corresponding to 127 mg/kg/day), and the LOAEL = 4500 ppm (corresponding to 391 mg/kg/day).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

391 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Guideline study

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January to April 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

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 obtained from Charles River Breeding Laboratories, MI, USA. They were acclimatised for approximately 3 weeks prior to study initiation. The rats were 8 weeks old at study initiation and weighed 221.3 - 264.5 g (males) and 166.7 - 219.11 g (females). Pretest health screens and viral screens were performed. Upon arrival the rats were housed in pairs in suspended stainless steel cages with mesh floors. After 1 week they were housed individually. Ground Purina Certified Rodent Chow #5002 and tap water were provided ad libitum. Temperature was maintained from 66-75°F and relative humidity was maintained from 40 to 70%. Lighting was provided on a 12 hour light/dark cycle, and there were at least 8 air changes per hour.
In life dates: 11 January 1988 to 12 April 1988.

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

The concentration of the test substance (expressed in terms of didecyldimethylammonium chloride; 80.8%) in the vehicle was 0, 0.1, 0.3 and 0.6% (w/w). Solutions were prepared weekly and stored at room temperature. Controls were dosed with water (the vehicle) at a constant dosing volume of 2.0 ml/kg bw.

Eight days prior to the first dose, the hair was clipped from the dorsal area of the trunk. All of the animals were wrapped in the manner used during the study for 6 hours/day for 4 days during the week prior to dosing. Animals that did not adapt to the pre-treatment wrapping procedure were not used in the study. Prior to the first dose, the fur was reclipped in preparation for dosing. Animals were reclipped in the afternoons as needed throughout the study, and on each Friday afternoon. The test material was applied directly to the back at each application. The entire application site was covered with a sterile 8-ply gauze pad and the rats were wrapped using Vetrap bandaging tape and secured with elastic tape. Dressings were left in place for approximately 6 hours. After dressing removal, the application site was rinsed with water and blotted using an 8-ply gauze pad. All animals were dosed 5 days/week, for 13 weeks. The females were also dosed on Saturday of the 13th week so that only 2 days elapsed between administration of the last dose and the final sacrifice.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity studies were performed on samples from all dosing solutions, and indicated that the test material was uniformly distributed. Stability studies indicated that the test material was stable in solution for at least 14 days. Concentration analyses ranged from 94.2 to 108.0% of nominal for all 3 concentrations.

Duration of treatment / exposure:

90 days

Frequency of treatment:

5 days/week; 6 hours/day (Monday through Friday)

Dose / conc.:

2 mg/kg bw/day (nominal)

Dose / conc.:

6 mg/kg bw/day (nominal)

Dose / conc.:

12 mg/kg bw/day (nominal)

No. of animals per sex per dose:

15/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Controls were exposed to the vehicle only (water). All solutions of the test substance were corrected for percentage active ingredient. The highest dose administered (0.6%) was chosen as the maximum dose that could be applied dermally without causing significant skin irritation. Only animals with an intact and normal epidermis were used in the study.

Positive control:

Not applicable.

Observations and examinations performed and frequency:

Animals were observed once a week for detailed clinical observations, and six days a week for overt clinical signs. Skin reactions were scored according to a modified Draize procedure when signs were observed on Friday after dosing and on Monday before dosing. Observations for mortality and moribundity were made twice daily. Bodyweight data and food consumption data were collected weekly for all animals. Ophthalmoscopic examinations took place prior to study initiation and prior to sacrifice. Blood samples were collected from all animals prior to sacrifice for haematology and clinical chemistry analyses. The following haematology parameters were evaluated: erythrocyte count, haemoglobin, haematocrit, erythrocyte indices, platelet count, total leukocyte count, differential leukocyte count, reticulocyte count. The following clinical chemistry parameters were determined: glucose, urea nitrogen, creatinine, AST (SGPT), ALT (SGOT), creatine kinase, gamma glutamyl transpeptidase, alkaline phosphatase, total protein, total cholesterol, albumin, globulin, A/G ratio, total bilirubin, direct bilirubin, indirect bilirubin, calcium, phosphorous, sodium, potassium, chloride.

Sacrifice and pathology:

All surviving rats were necropsied and examined for gross pathologic changes. The following tissues were processed for histopathology: gross lesions, spinal cord, brain, pituitary, thyroid, thymic region, trachea, lungs, heart, salivary gland, liver, spleen, kidneys, adrenals, pancreas, testes, epididymides, prostate, seminal vesicles, ovaries, uterus, vagina, mammary gland, skin, oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, urinary bladder, lymph nodes, sciatic nerve, sternum, femur, skeletal muscle, eyes, aorta, exorbital lacrymal gland. Histopathologic examinations were performed on the harvested tissues from all animals in the control and high dose groups. In addition, the skin, lungs, liver, kidneys and all gross lesions were processed and examined histologically from all animals in the mid and low dose groups. Organ weights were obtained for the liver, kidneys, spleen, heart, brain with stem, adrenals, testes, ovaries.

Other examinations:

No other examinations reported.

Statistics:

Parametric variables were intercompared for the dose and control groups using Levene’s test for homogeneity of variances, by analysis of variance and by pooled variance t-tests. Non-parametric data were analyzed by the Kruskal-Wallis test or by the Wilcoxon rank sum test as modified by Mann-Whitney. Frequency data were compared using Fisher’s exact tests.

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

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:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Details on results:

MORTALITY
One female each from the low and high dose groups died during the study on Days 77 and 63 respectively, and one female from the mid dose groups was sacrificed moribund on Day 75. These deaths were not considered related to treatment.

CLINICAL SIGNS
No treatment-related signs were observed in either sex at any dose throughout the 90-day dosing period. Skin irritation (erythema, oedema) was observed at 6 and 12 mg a.s./kg/d primarily early in the study (Days 5-8).

BODY WEIGHT
No treatment-related effects in either sex at any dose.

FOOD CONSUMPTION
No treatment-related effects in either sex at any dose.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related effects in either sex at any dose.

HAEMATOLOGY
No treatment-related effects in either sex at any dose.

CLINICAL CHEMISTRY
No treatment-related effects in either sex at any dose.

ORGAN WEIGHTS
No treatment-related effects in either sex at any dose. Statistically significant differences (adrenal glands relative to body weight in males from the mid dose group and spleen weight relative to body weight in females from the mid dose group) were considered spurious.

GROSS PATHOLOGY
Treatment-related gross findings indicative of minimal to mild skin irritation were observed at the two highest doses.

HISTOPATHOLOGY: NON-NEOPLASTIC
Micropscopically, lesions wer eobserved in the treated skin for some animals from each treatment level. The greatest incidence of animals with clear histologic evidence of dermal and/or epidermal inflammation (epidermitis, dermatitis, vacuolar degeneration, haemorrhage, ulceration) occurred for females at the high dose. No other treatment-related observations were recorded.

Key result

Dose descriptor:

NOAEL

Effect level:

12 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Equivalent to 14.9 mg test substance/kg bw/d. No treatment-related effects were observed at any dose.

Key result

Critical effects observed:

not specified

No further information available.

Conclusions:

The NOAEL was considered to be 12 mg a.s./kg bw/d (equivalent to 14.9 mg test substance/kg bw/d), the highest dose tested.

Executive summary:

Male and female Sprague-Dawley rats were exposed dermally to Bardac 2280 (Didecyldimethylammonium Chloride aqueous/alcohol solution) for 90 days, according to USEPA OPP 82 -3 and OECD 411. The test substance was applied at 0, 2, 6 and 12 mg a.s./kg bw/d under occlusive dressings for 6 hours/day, 5 days/week for 90 days. The rats were observed for clinical signs and mortality and signs of dermal irritation. Bodyweights and food consumption were recorded weekly. At study termination ophthalmoscopic examinations, haematology and clinical chemistry analyses, gross necropsy and histopathology were carried out.

Other than a brief period of skin irritation at the two highest doses observed early in the study (Day 5 -8), and gross and microscopic indication of skin irritation after 90-days of treatment, no effects from repeated dermal exposure to the test substance were observed. The NOAEL was considered to be 12 mg a.s./kg bw/d, the highest dose tested (equivalent to 14.9 mg test substance/kg bw/d) .

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

14.9 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

A high quallity guideline- and GLP-compliant study is available.

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January to April 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

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 obtained from Charles River Breeding Laboratories, MI, USA. They were acclimatised for approximately 3 weeks prior to study initiation. The rats were 8 weeks old at study initiation and weighed 221.3 - 264.5 g (males) and 166.7 - 219.11 g (females). Pretest health screens and viral screens were performed. Upon arrival the rats were housed in pairs in suspended stainless steel cages with mesh floors. After 1 week they were housed individually. Ground Purina Certified Rodent Chow #5002 and tap water were provided ad libitum. Temperature was maintained from 66-75°F and relative humidity was maintained from 40 to 70%. Lighting was provided on a 12 hour light/dark cycle, and there were at least 8 air changes per hour.
In life dates: 11 January 1988 to 12 April 1988.

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

The concentration of the test substance (expressed in terms of didecyldimethylammonium chloride; 80.8%) in the vehicle was 0, 0.1, 0.3 and 0.6% (w/w). Solutions were prepared weekly and stored at room temperature. Controls were dosed with water (the vehicle) at a constant dosing volume of 2.0 ml/kg bw.

Eight days prior to the first dose, the hair was clipped from the dorsal area of the trunk. All of the animals were wrapped in the manner used during the study for 6 hours/day for 4 days during the week prior to dosing. Animals that did not adapt to the pre-treatment wrapping procedure were not used in the study. Prior to the first dose, the fur was reclipped in preparation for dosing. Animals were reclipped in the afternoons as needed throughout the study, and on each Friday afternoon. The test material was applied directly to the back at each application. The entire application site was covered with a sterile 8-ply gauze pad and the rats were wrapped using Vetrap bandaging tape and secured with elastic tape. Dressings were left in place for approximately 6 hours. After dressing removal, the application site was rinsed with water and blotted using an 8-ply gauze pad. All animals were dosed 5 days/week, for 13 weeks. The females were also dosed on Saturday of the 13th week so that only 2 days elapsed between administration of the last dose and the final sacrifice.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity studies were performed on samples from all dosing solutions, and indicated that the test material was uniformly distributed. Stability studies indicated that the test material was stable in solution for at least 14 days. Concentration analyses ranged from 94.2 to 108.0% of nominal for all 3 concentrations.

Duration of treatment / exposure:

90 days

Frequency of treatment:

5 days/week; 6 hours/day (Monday through Friday)

Dose / conc.:

2 mg/kg bw/day (nominal)

Dose / conc.:

6 mg/kg bw/day (nominal)

Dose / conc.:

12 mg/kg bw/day (nominal)

No. of animals per sex per dose:

15/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Controls were exposed to the vehicle only (water). All solutions of the test substance were corrected for percentage active ingredient. The highest dose administered (0.6%) was chosen as the maximum dose that could be applied dermally without causing significant skin irritation. Only animals with an intact and normal epidermis were used in the study.

Positive control:

Not applicable.

Observations and examinations performed and frequency:

Animals were observed once a week for detailed clinical observations, and six days a week for overt clinical signs. Skin reactions were scored according to a modified Draize procedure when signs were observed on Friday after dosing and on Monday before dosing. Observations for mortality and moribundity were made twice daily. Bodyweight data and food consumption data were collected weekly for all animals. Ophthalmoscopic examinations took place prior to study initiation and prior to sacrifice. Blood samples were collected from all animals prior to sacrifice for haematology and clinical chemistry analyses. The following haematology parameters were evaluated: erythrocyte count, haemoglobin, haematocrit, erythrocyte indices, platelet count, total leukocyte count, differential leukocyte count, reticulocyte count. The following clinical chemistry parameters were determined: glucose, urea nitrogen, creatinine, AST (SGPT), ALT (SGOT), creatine kinase, gamma glutamyl transpeptidase, alkaline phosphatase, total protein, total cholesterol, albumin, globulin, A/G ratio, total bilirubin, direct bilirubin, indirect bilirubin, calcium, phosphorous, sodium, potassium, chloride.

Sacrifice and pathology:

All surviving rats were necropsied and examined for gross pathologic changes. The following tissues were processed for histopathology: gross lesions, spinal cord, brain, pituitary, thyroid, thymic region, trachea, lungs, heart, salivary gland, liver, spleen, kidneys, adrenals, pancreas, testes, epididymides, prostate, seminal vesicles, ovaries, uterus, vagina, mammary gland, skin, oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, urinary bladder, lymph nodes, sciatic nerve, sternum, femur, skeletal muscle, eyes, aorta, exorbital lacrymal gland. Histopathologic examinations were performed on the harvested tissues from all animals in the control and high dose groups. In addition, the skin, lungs, liver, kidneys and all gross lesions were processed and examined histologically from all animals in the mid and low dose groups. Organ weights were obtained for the liver, kidneys, spleen, heart, brain with stem, adrenals, testes, ovaries.

Other examinations:

No other examinations reported.

Statistics:

Parametric variables were intercompared for the dose and control groups using Levene’s test for homogeneity of variances, by analysis of variance and by pooled variance t-tests. Non-parametric data were analyzed by the Kruskal-Wallis test or by the Wilcoxon rank sum test as modified by Mann-Whitney. Frequency data were compared using Fisher’s exact tests.

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

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:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Details on results:

MORTALITY
One female each from the low and high dose groups died during the study on Days 77 and 63 respectively, and one female from the mid dose groups was sacrificed moribund on Day 75. These deaths were not considered related to treatment.

CLINICAL SIGNS
No treatment-related signs were observed in either sex at any dose throughout the 90-day dosing period. Skin irritation (erythema, oedema) was observed at 6 and 12 mg a.s./kg/d primarily early in the study (Days 5-8).

BODY WEIGHT
No treatment-related effects in either sex at any dose.

FOOD CONSUMPTION
No treatment-related effects in either sex at any dose.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related effects in either sex at any dose.

HAEMATOLOGY
No treatment-related effects in either sex at any dose.

CLINICAL CHEMISTRY
No treatment-related effects in either sex at any dose.

ORGAN WEIGHTS
No treatment-related effects in either sex at any dose. Statistically significant differences (adrenal glands relative to body weight in males from the mid dose group and spleen weight relative to body weight in females from the mid dose group) were considered spurious.

GROSS PATHOLOGY
Treatment-related gross findings indicative of minimal to mild skin irritation were observed at the two highest doses.

HISTOPATHOLOGY: NON-NEOPLASTIC
Micropscopically, lesions wer eobserved in the treated skin for some animals from each treatment level. The greatest incidence of animals with clear histologic evidence of dermal and/or epidermal inflammation (epidermitis, dermatitis, vacuolar degeneration, haemorrhage, ulceration) occurred for females at the high dose. No other treatment-related observations were recorded.

Key result

Dose descriptor:

NOAEL

Effect level:

12 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Equivalent to 14.9 mg test substance/kg bw/d. No treatment-related effects were observed at any dose.

Key result

Critical effects observed:

not specified

No further information available.

Conclusions:

The NOAEL was considered to be 12 mg a.s./kg bw/d (equivalent to 14.9 mg test substance/kg bw/d), the highest dose tested.

Executive summary:

Male and female Sprague-Dawley rats were exposed dermally to Bardac 2280 (Didecyldimethylammonium Chloride aqueous/alcohol solution) for 90 days, according to USEPA OPP 82 -3 and OECD 411. The test substance was applied at 0, 2, 6 and 12 mg a.s./kg bw/d under occlusive dressings for 6 hours/day, 5 days/week for 90 days. The rats were observed for clinical signs and mortality and signs of dermal irritation. Bodyweights and food consumption were recorded weekly. At study termination ophthalmoscopic examinations, haematology and clinical chemistry analyses, gross necropsy and histopathology were carried out.

Other than a brief period of skin irritation at the two highest doses observed early in the study (Day 5 -8), and gross and microscopic indication of skin irritation after 90-days of treatment, no effects from repeated dermal exposure to the test substance were observed. The NOAEL was considered to be 12 mg a.s./kg bw/d, the highest dose tested (equivalent to 14.9 mg test substance/kg bw/d) .

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

Study duration:

subchronic

Species:

rat

Quality of whole database:

A high quallity guideline- and GLP-compliant study is available.

Additional information

Some of the data summarised in this section are based on Didecyldimethylammonium Chloride (Bardac 2280), a chemical and structural analogue of N,N-Didecyl-N-methyl-poly(oxyethyl)ammonium Propionate. 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.

Subchronic toxicity - N,N-Didecyl-N-methyl-poly(oxyethyl)ammonium Propionate

Administration of the test substance in the diet over 90-days did not result in the death of rats (Thomas et al, 1999). No clinically observable signs of toxicity were detected. However at the highest dietary administration dose level (391 mg/kg bw/day), evident toxicity was noted in rats; reduction in body weight gain, food consumption, clinical chemistry changes, small spleen in females, reduced absolute liver weight and body weight relative liver weight. The NOEL was equivalent to 127 mg/kg bw/day and the NOAEL was 391 mg/kg bw/day (Thomas et al, 1999).

Subchronic toxicity - Didecyldimethylammonium Chloride

Bardac 2280 was administered orally (gavage) to Beagle dogs for 8 weeks. Administration induced emesis, salivation, few or no faeces, lacrimation and thin appearance, but induced a very low level of mortality since only two deaths (one animal from each sex) were observed at the highest dose level. The NOAEL was 30 mg/kg bw/d (Osheroff, 1990).

The dermal application of Bardac 2280 to rats for a period of 90 days induced a brief period of skin irritation at the two highest doses early in the study, however no effects from the repeated dermal exposure were observed. The NOAEL was equal to 12 mg/kg bw/day; equivalent to 14.9 mg test substance/kg bw/d (Gill and Van Miller, 1988).

Chronic toxicity - Didecyldimethylammonium Chloride

Chronic toxicity studies with N,N-Didecyl-N-methyl-poly(oxyethyl)ammonium Propionate have not been conducted in dogs or rats, therefore data from the analogue compound Didecyldimethylammonium Chloride can be used in order to estimate the behaviour of N,N-Didecyl-N-methyl-poly(oxyethyl)ammonium Propionate when administered orally for a period of one or two years.

 

Oral (gavage) administration of Bardac 2280 to dogs for 52 weeks resulted in minimal changes in red cell parameters and serum protein determinations. When administered at the two highest doses, 10 or 20 mg a.s./kg bw/d resulted in g.i.-related complications emesis, salivation and also softened stool was noted. The NOAEL was considered to be 10 mg a.s./kg bw/d (equivalent to 12.4 mg/kg bw/d test substance) and the LOAEL was 20 mg a.s./kg bw/day (equivalent to 24.8 mg/kg bw/d test substance) (Schulze, 1991).

 

Bardac 2280 was administered to rats in the diet for 104 weeks. The highest dietary concentration resulted in decreases in food consumption, body weights, and an increased incidence of bile duct hyperplasia and blood in the sinuses of mesenteric lymph nodes for both sexes. The NOAEL was considered to be 32 and 41 mg a.s./kg bw/d for males and females respectively (equivalent to 39.6 and 50.7 mg test substance/kg bw/d for males and females, respectively) (Gill et al, 1991).

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study provides the lowest endpoint

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
N,N-Didecyl-N-methyl-poly(oxyethyl)ammonium Propionate is not volatile as the vapour pressure is 1.8E-6 Pa2. N,N-Didecyl-N-methyl-poly(oxyethyl)ammonium Propionate is sold as an aqueous dilution and therefore does not contain respirable particles. In addition, the results of the skin irritation study indicate that the substance is corrosive. Thus, it is considered that a short term repeated dose inhalation toxicity study is not justified.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Only one study is available for this endpoint

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Only one study is available for this endpoint

Justification for classification or non-classification

Based on the available data classification according to Regulation (EC) No 1272/2008 is not required.