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

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

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

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

bis(decyl)(2-hydroxyethyl)methylazanium propanoate

EC Number:

619-057-3

Cas Number:

94667-33-1

Molecular formula:

C29 H62 N O4 . C3 H5 O2

IUPAC Name:

bis(decyl)(2-hydroxyethyl)methylazanium propanoate

Constituent 2

Reference substance name:

alpha.-[2-(Didecylmethylammonio)ethyl]-.omega.-hydroxy-poly(oxy-1,2-ethanediyl) propionate

IUPAC Name:

alpha.-[2-(Didecylmethylammonio)ethyl]-.omega.-hydroxy-poly(oxy-1,2-ethanediyl) propionate

Constituent 3

Reference substance name:

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

IUPAC Name:

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

Constituent 4

Reference substance name:

Bardap 26

IUPAC Name:

Bardap 26

Details on test material:

Bardap 26: a colourless, slightly viscous liquid (referred to as P4140 in the study report). The test substance was stored at room temperature in the dark.

Test animals

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.

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

42 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

127 mg/kg bw/day (nominal)

Doses / concentrations 3

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.

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

1

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

2

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

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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).