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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008 - 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according OECD guidance 422.
Justification for type of information:
Read-across data is presented from quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808-53-2, EC Number 276-038-9. This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3650
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Quaternary ammonium compounds, coco alkylbis(hydroxyethyl)methyl, chlorides
EC Number:
274-846-6
EC Name:
Quaternary ammonium compounds, coco alkylbis(hydroxyethyl)methyl, chlorides
Cas Number:
70750-47-9
IUPAC Name:
Quaternary ammonium compounds, coco alkylbis(hydroxyethy​l)​methyl, chlorides
Specific details on test material used for the study:
- Name of test material (as cited in study report): Quarternary ammonium compounds, coco alkylmethylbis(hydroxyethyl)methyl, chlorides
- Physical state: Liquid
- Analytical purity: 75 %
- Lot/batch No.: 7855 19
- Expiration date of the lot/batch: August 2008
- Storage condition of test material: Room temperature (20 ± 5 °C)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd
- Age at study initiation: 11 weeks
- Weight at study initiation: 292 to 326g (males) 178 to 212g (females)
- Fasting period before study:
- Housing: Makrolon type-3 cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 (°C)
- Humidity: 30-70 %
- Air changes: 10-15 per hr
- Photoperiod: (12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Indivdual samples which were analysed ranged from 90.3 to 117.2% of nominal values (within the 20% acceptance limit).
Duration of treatment / exposure:
28-days minimum for males
Approximately 7 weeks for females
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
Basis:
other: gavage administration
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Basis:
other: gavage administration
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Basis:
other: gavage administration
No. of animals per sex per dose:
10 males and 10 females per dose group
Control animals:
yes
Details on study design:
- Dose selection rationale: Dose range finding study using dose levels of 20, 60 and 180 mg/kg/day resulting to be lethal at 180 mg/kg/day and in NOEL of 20 mg/kg/day
- Rationale for animal assignment: random

Control animals:
The control group was treated with a solution of Isopropanol anhydrous (CAS No. 00067-63-0) in water at the same dilution as for the highest dose level formulation.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGESIDE OBSERVATIONS:
Performed daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: once prior to the first administration of the test substance and weekly thereafter.

BODY WEIGHT:
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION:
- Food consumption for males: recorded weekly during and after pairing period
- Food consumption for females: pre-pairing period days 1-8 and 8-14; gestation days 0-7, 7-14 and 14 to 21 post coitum, and days 1-4 post partum

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood:
males - samples were collected on the day before or on the daz of sheduled necropsy.
females - samples were collected from lactating females 5 days post partum.
- Anaesthetic used for blood collection: light isoflurane anesthesia
- Animals fasted: 18 hours before collection
- How many animals: 5 male and 5 females
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood:
males - samples were collected on the day before or on the daz of sheduled necropsy.
females - samples were collected from lactating females 5 days post partum.
- Anaesthetic used for blood collection: light isoflurane anesthesia
- Animals fasted: 18 hours before collection
- How many animals: 5 male and 5 females
- Parameters checked in table [No.2] were examined.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: P-generation males and females were studied before the scheduled sacrifice and on days 3 or 4 post partum, respectively
- Dose groups that were examined: 5 animals per sex per group
- Battery of functions tested: cage side observations/ hand-held observations/ grip strength / rearing behaviou/reflexes /landing foot splay

Sacrifice and pathology:
NECROPSY: The following tissue samples were collected; prostate, seminal vesicles with coagulating gland, testes, epididymides, ovaries, brain, spinal chord, small and large intestines, stomach, liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea and lungs, uterus, urinary bladder, lymph nodes, sciatic nerve, bone marrow.
GROSS PATHOLOGY: Yes (see table 3)
HISTOPATHOLOGY: all tissues and organs collected at necropsy were examined by histopathlogical techniques.
Statistics:
The Dunnett-test.(manyy to one t-test= based on pooled variance estimate was applied if the variable could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.

The Steel-test (many.one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.

Fisher's exact-test was applied to macroscopic findings.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 100mg/kg bw/day one male and one female were found dead on day 5 of the pre-paring period. At 50 mg/kg bw/day one female was found dead on dead and 3 others were sacrificed due to ethical reasons. The 3 sacrificed females were observed to have ruffled fur, diarrhea and sedation 2 days before sacrfice on day 8 of pre-pairing period.
Mortality:
mortality observed, treatment-related
Description (incidence):
At 100mg/kg bw/day one male and one female were found dead on day 5 of the pre-paring period. At 50 mg/kg bw/day one female was found dead on dead and 3 others were sacrificed due to ethical reasons. The 3 sacrificed females were observed to have ruffled fur, diarrhea and sedation 2 days before sacrifice on day 8 of pre-pairing period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significant reduction in body weight gain in pre-pairing period in the 50mg/kg bw/day dose group
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 25 and 50 mg/kg bw/day the activity of alanine aminotransferase was dose dependently and statisically significantly increased. This was not accompanied by histopathological findings in the liver.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Effects on locomotor activity were observed in the assessment on low beam counts in an activity monitor. this was considered to be within the range of the historical control data.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Males: Thymus with foci in one male in the 50 mg/kg bw/day; mucosa of forestomach with several nodules in one male at 25mg/kg bw/day.

Females: Yellowish mucosa of the forestomach in one female at 50 mg/kg bw/day.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Marked inflammatory lesions in the forestomach observed in animals in the 50 and 100 mg/kg bw/day which resulted in death.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
At 100mg/kg bw/day one male and one female were found dead on day 5 of the pre-paring period. At 50 mg/kg bw/day one female was found dead on dead and 3 others were sacrificed due to ethical reasons. The 3 sacrificed females were observed to have ruffled fur, diarrhea and sedation 2 days before sacrfice on day 8 of pre-pairing period.

BODY WEIGHT AND WEIGHT GAIN
Significant reduction in body weight gain in pre-pairing period in the 50mg/kg bw/day dose group.

HAEMATOLOGY
No test item-related effects were noted. In group 3, the statistically singificantly high level of abolute neutrophils count (+42.5% compared to control group) was within the range of the historical reference values.

CLINICAL CHEMISTRY
Males: At 25 and 50 mg/kg bw/day the activity of alanine aminotransferase was dose dependently and statisically significantly increased. This was not accompanied by histopathological findings in the liver. Other effects were observed in 25 and 50 mg/kg bw/day dose groups but were within the historical reference values
Females: 50 mg/kg bw/day the activity of creatine kinase wasstatistically significantly increased (+38.6 versus the control group); the levels of calcium and phosphorus were statistically significantly increased (+5.6% and 22.1% versus the control group, respectively). All these values were within the range of the historical control reference values.

NEUROBEHAVIOUR
Effects on locomotor activity were observed in the assessment on low beam counts in an activity monitor. this was considered to be within the range of the historical control data

ORGAN WEIGHTS
No effects on organ weight

GROSS PATHOLOGY
Males: Thymus with foci in one male in the 50 mg/kg bw/day; mucosa of forestomach with several nodules in one male at 25mg/kg bw/day.
Females: Yellowish mucosa of the forestomach in one female at 50 mg/kg bw/day.

HISTOPATHOLOGY: NON-NEOPLASTIC
Marked inflmamatory lesions in forestomach of animals in 50 and 100 mg/kg bw/day dose group were obsereved after sacrifice.
Forestomach erosion was noted in one male at 100mg/kg bw/day.
Hyperkeratosis/acanthosis of forestomach mucosa was noted in 5 males and 5 females at 25 mg/kg/day, in 5 males and 6 females at 50 mg/kg bw/day, and in 2 males and 6 females at 100 mg/kg bw/day.

The changes in the forestomach were considered to be a local effect resulting from the administation of a irritant substance by oral gavage.

Effect levels

Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: general toxicity

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
According to the study report, no general NOAEL could be established in this study as moderate actute/subacute necrotizing inflammation of the forestomach mucosa was observed in 1 out 5 females examined at 25 mg/kg/day, the lowest dose tested, and increased activity of alanine aminotransferase was obsereved in males at 25 and 50 mg/kg/day.

The general NOAEL is therefore set to 25 mg/kg body weight/day for this substance.
Executive summary:

The repeated dose toxicity of the test substance, Quaternary ammonium compounds, coco alkylbis(hydroxyethyl)methyl chlorides, CAS Number 70750 -47 -9, EC Number 274-846-6, [Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808 -53 -2, EC Number 276 -038 -9], was investigated in a study conducted according to OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) / OPPTS 870.3650.

This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Under the conditions of thus study the general NOAEL was determined to be 25 mg/kg body weight/day for this substance.

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