Bis(hydroxyethyl)methyloleylammonium chloride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The toxicity to reproduction 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) / EPA 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 the study the NOAEL for reproductive/developmental toxicity was set at the 25mg/kg bw/day dose level. However, considering that there were no reproductive effects in the surviving dams treated witih 50 mg/kg bw/day, and no changes in the male reproductive system were noted, the NOAEL may be even higher.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008 - 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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.

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: EPA OPPTS 870.3650

Deviations:

no

GLP compliance:

yes

Limit test:

no

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)

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat, HanRcc: WIST(SPF)

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)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

28-days minimum for males
Approximately 7 weeks for females

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation:until copulation was observed
- Proof of pregnancy: vaginal plug / sperm in vaginal smear
- After successful mating each pregnant female was caged individually

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

Dose / conc.:

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

Remarks:

Vehicle control (23% isopropanol in water) dose voolume 10mL/kg

Dose / conc.:

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

Remarks:

Tests substance contains 23% ispopropanol dose adjusted for 75% purity dose volume 10mL/kg

Dose / conc.:

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

Remarks:

Tests substance contains 23% ispopropanol dose adjusted for 75% purity dose volume 10mL/kg

Dose / conc.:

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

Remarks:

Tests substance contains 23% ispopropanol dose adjusted for 75% purity dose volume 10mL/kg

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.

Parental animals: Observations and examinations:

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.2] 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.3] 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

Oestrous cyclicity (parental animals):

not recorded

Sperm parameters (parental animals):

Parameters examined in P0 male parental generations:
Testis weight, epididymis weight, sperm staging

Litter observations:

Litters were examined for litter size, live births, still births and gross anomalies. The sex ratios of the pups were recorded. Pups were weighed individually (without identification) on days 0, 1 and 4 post partum.

Postmortem examinations (parental animals):

NECROPSY: The following tissue samples were collected; ovaries, prostate, seminal vesicles with coagulating gland, testes, epididymides, 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
HISTOPATHOLOGY: all tissues and organs collected at necropsy were examined by histopathlogical techniques.

Postmortem examinations (offspring):

Examined for gross anomilies

Statistics:

The Dunnett-test (many 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.

Reproductive indices:

Successful breeding pairs, fertility index and conception rate.

Offspring viability indices:

The offspring the viability index was 96.4, 99.3 and 100.0%, in order of ascending dosages. (0, 25 and 50 mg/kg respectively).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 100mg/kg bw/day one male and two females were found dead on day 5 of the pre-paring period, the remaining animals were terminated for ethical reasons. At 50 mg/kg bw/day one female was found dead on day 6 and 4 others were sacrificed due to ethical reasons on day 8.

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

At 100mg/kg bw/day one male and two females were found dead on day 5 of the pre-paring period, the remaining animals were terminated for ethical reasons. At 50 mg/kg bw/day one female was found dead on day 6 and 4 others were sacrificed due to ethical reasons on day 8.

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):

effects observed, treatment-related

Description (incidence and severity):

Males: at 25 and 50 mg/kg/day mean food consumption was dose-dependently reduced during the first part of the pre-pairing period. Afterwards, no test item-related effects were noted. Females: at 50 mg/kg/day mean food consumption was reduced during the first part of the pre-pairing period. Afterwards, no test item-related effects were noted.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males
No test item—related effects were noted. In group 3, the statistically significantly high level of absolute neutrophilis count (+42.5% compared to the control group) was within the range of the historical reference values.

In groups 2 no altered parameters were observed.

Females
In group 2, the relative count of monocytes was statistically significantly higher (+2l.7% compared to the control group value). This was not considered to be a test item—related effect since the absolute count was not significantly increased and the higher levels of relative and absolute counts of monocytes also observed in group 3 were not statistically significant.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males
In groups 2 and 3, the activity of alanine aminotransferase was dose dependently and statistically significantly increased (+68.7% and +96.8% compared to the control group, respectively). These increases were not accompanied by histopathological findings in the liver.
In group 3, the statistically significantly lower level of total bilirubin and phosphorus (-39.0% and -12.5% versus the control, respectively) were within the range of the reference values. In group 2 the statistically significantly higher levels of cholesterol and sodium (+20.0% and +0.8% compared to the control group values, respectively) were within the range of the historical reference values.

Females
In group 3, the activity of creatine kinase was statistically significantly increased (+38.6% versus the control group); the levels of calcium and phosphorus were statistically significantly increased (+5.6% and +22.l% versus the control group, respectively) and the level of chloride was statistically significantly decreased (-3.0% versus the control group). All these values were within the range of historical control reference values. The activity of alanine aminotransferase was statistically significantly increased (+75.0% versus the control group) and was not accompanied by histopathological findings in the liver.

In groups 2 and 3, the levels of creatine were statistically significantly decreased (-12.6% and - 16.5% compared to the control group, respectively) but within the range of historical control reference values and therefore not considered to be of toxicological relevance.

In group 2, the statistically significantly higher level of calcium (+6.0% compared to control group) was within the range of historical control reference values.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

All animals in group 3 were noted to have ruffled fur during the open field observations. This sign was not considered indicative of any neurological effect, but related to the general poor health status of these animals.

The other parameters such as rearing number and puddles of urine, activity and salivation did not give any indication of a test item-related effect. Common findings such as fearfulness and spontaneous vocalization when the rat was removed from the cage were observed in a single male in the control group.

Mean values of grip strength (fore and hind paws) and landing foot splay gave no indication of test item-related effects. Body temperature in males was statistically significantly lower in group 2 compared to the control group (37.6 °C compared to 38.3 °C in the control group). Since the body temperature in group 3 was not affected this finding was considered to be incidental.

Locomotor activity was assessed quantitatively in terms of low beam counts in an activity monitor. For females in group 3, the locomotor activity was statistically significantly lower during the last 6 minutes of the 30 minutes measurement period, which was considered to be of incidental nature since it was within the range of historical control data. No other statistical significances occurred.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

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.

Acute/subacute necrotizing inflammation of the forestomach mucosa was diagnosed in one female in group 2 (moderate), one female in group 3 (slight), and ten males and nine females in group 4 (slight to marked in degree).

Forestomach erosion was noted in one male in group 4.

Hyperkeratosis/acanthosis of forestomach mucosa was noted in five males and five females in group 2 (minimal to marked in degree), five males and six females in group 3 (moderate to marked in degree), and two males and six females in group 4 (slight to moderate in degree). The changes in the stomach were considered to be a local effect consequent to the highly irritating properties of the test item administered as a bolus of gavage

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Under the conditions of this study, the test item did not reveal effects on the completeness of stages or cell populations in any of the treated groups, compared to the control. There was no indication of maturation arrest, re-absorption of sperm or any other degeneration type.

Reproductive performance:

no effects observed

Description (incidence and severity):

Fertility index and conception rate were 100% at 0, 25 and 50 mg/kg/day.

Fertility index and conception rate were 100% at 0, 25 and 50 mg/kg/day.
Mean number of corpora lutea per dam was similar in all groups.
The mean duration of gestation was unaffected by exposure to the test substance.
The mean number of implantations per dam was similar in all groups. The total and mean post implnatation loss was not affected by the treatment with the test item.

Key result

Dose descriptor:

NOAEL

Remarks:

general

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Moderate necrotizing inflammation of the forestomach mucosa in one out of the five females examined at 25 mg/kg/day, however this is a local effect so it does not prevent the setting of a systemic NOAEL.

Key result

Dose descriptor:

NOEL

Remarks:

reproduction

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Considering that there were no reproductive effects in the surviving dams treated with 50 mg/kg/day, and no changes in the male reproductive system were noted, this NOEL may be even higher.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

duodenum

ileum

jejunum

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormal findings were observed during the macroscopic examination of F1 pups

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Litter size at first litter check was not affected by the treatment with the test item. Post natal loss was not affected by the treatment of the test substance.

Key result

Dose descriptor:

NOEL

Remarks:

developmental

Generation:

F1

Effect level:

> 25 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Could be higher as no adverse effects were seen at 50mg/kg, but only 5 parental females survived against 8 required.

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

yes

Reproduction data. summary of performance

Group (mg/kg/day)	1 (0)	2 (25)	3 (50)	4 (100)
Female numbers	41 -50	51 -60	61 -70	71 -80
No. of females paired	10	10	5	-
No. females died before scheduled necropsy	-	-	5	10
No. females with early delivery	1	0	0	-
No. females that reared pups until day 4 post partum	10	10	5

Conclusions:

The NOAEL for reproductive/developmental toxicity was set at the 25mg/kg bw/day dose level. Considering that there were no reproductive effects in the surviving dams treated witih 50 mg/kg bw/day, and no changes in the male reproductive system were noted, the NOAEL may be even higher.

Executive summary:

The toxicity to reproduction 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) / EPA 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 the study the NOAEL for reproductive/developmental toxicity was set at the 25mg/kg bw/day dose level. However, considering that there were no reproductive effects in the surviving dams treated witih 50 mg/kg bw/day, and no changes in the male reproductive system were noted, the NOAEL may be even higher.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015 - 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Specific details on test material used for the study:

Identification : Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides (CAS No 71808-53-2)
Physical State/Appearance : Dark yellow liquid
Chemical Name : C12-18-alkylbis(hydroxyethyl)methyl, chloride
Purity : 75%
Batch Number : 1261747
Label : Quaternary ammonium compounds, C12-18-, alkylbis
(hydroxyethyl) methyl, chlorides 7302761 1261747
Expiry 2016-01-16
Date Received : 04 April 2015
Storage Conditions : Store in cool place. Prevent oxidation. Keep in wellventilated
place. Room temperature in the dark.
Expiry Date : 16 January 2016

A correction for purity was made.

For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in distilled water. The stability and homogeneity (by visual inspection) of the test item formulations were determined by Envigo Research Limited, Shardlow, UK Analytical Services. Formulations were therefore prepared weekly and stored at approximately +4 °C in the dark.

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 169 to 260g.

The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly mean temperatures and humidity are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20% respectively; short term
deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.

The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test item was administered daily, from Day 3 to Day 19 of gestation, by gavage. Control animals were treated in an identical manner with the vehicle alone.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken on two occasions of each test item formulation and were analyzed for concentration of Quaternary ammonium compounds, C12-18-alkylbis (hydroxyethyl)methyl, chlorides (CAS No 71808-53-2) at Envigo Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within ± 3% of the nominal concentration.

Details on mating procedure:

Animals were time-mated and delivered prior to day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation.

Duration of treatment / exposure:

The test item was administered daily, from Day 3 to Day 19 of gestation, by gavage. Control animals were treated in an identical manner with the vehicle alone.

Frequency of treatment:

Daily

Duration of test:

19 days

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels 5, 15, and 25 mg/kg bw/day A.I.(active ingredient; incorporating a correction factor for 75% purity of the test item). A further group of twenty-four time mated females was exposed to the vehicle only (Distilled water) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study.

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weight, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Maternal examinations:

Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were performed immediately before and soon after dosing and one hour post dosing. All observations were recorded.

Individual body weights were recorded on Day 3 and on Days 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

Water intake was observed daily by visual inspection of the water bottles for any overt changes.

Ovaries and uterine content:

All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination. The ovaries and uteri of pregnant females were removed, examined and the following data recorded:

i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight

The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.

Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes

All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):

Left Horn Cervix Right Horn
L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16

V = viable fetus

Fetal examinations:

The fetuses were killed by subcutaneous injection of a suitable barbiturate. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.

Statistics:

All data was summarized in tabular form, including reproductive indices. Group mean values were calculated to include data from all females with live fetuses on Day 20 of gestation. Values given in appendices may represent rounded values for presentation purposes. Group mean values were generally calculated using unrounded values therefore it is not always possible to calculate the exact group mean values from values presented in the appendices.

As the litter was standard unit of assessment, values were first calculated within the litter and group mean values represent the mean of these individual litter values.

Pre and Post Implantation Loss
Percentage pre-implantation loss was calculated as:

number of corpora lutea - number of implantations/number of corpora lutea x 100

Percentage post-implantation loss was calculated as:

number of implantations - number of live fetuses/number of implantations x 100

Sex Ratio
Sex ratio was calculated as:
% male fetuses (sex ratio) = Number of male fetuses / Total number of fetuses x 100

Statistical Analysis
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test. All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.

Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis nonparametric analysis of variance and Mann-Whitney ‘U’ test.
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p≥0.05 (not significant)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were detected.
Two females treated with 25 mg/kg bw/day A.I. showed clinical signs of noisy respiration between Days 15 to 18 (female 84) and Days 13 to 17 (female 93). This may be related to the irritant nature of the test item, but as it was not associated with any other detrimental findings on the study it was deemed to be of no toxicological importance.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

There was no unscheduled deaths.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no adverse effects evident in body weight development for females treated with 5, 15 or 25 mg/kg bw/day A.I.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no adverse effects on food consumption for females treated with 5, 15 or 25 mg/kg bw/day A.I.

Food efficiency:

not specified

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

no effects observed

Description (incidence and severity):

Daily visual inspection of water bottles did not reveal any overt intergroup differences

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic abnormalities were detected for any of the females.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Number of abortions:

not specified

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

One female treated with 15 mg/kg bw/day showed no live fetuses on Day 20 of gestation, this female had 100% post implantation loss. This finding was considered to be incidental.

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

not specified

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in the uterine parameters examined, in fetal viability or growth and development.

There were six non pregnant females in total (2 controls, 2 treated with 5 mg/kg bw/day A.I. and 2 treated with 15 mg/kg bw/day A.I.).

Key result

Dose descriptor:

NOEL

Effect level:

25 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No treatment related effects

Fetal body weight changes:

not specified

Reduction in number of live offspring:

not specified

Changes in sex ratio:

not specified

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal weights across the test item-treated dose groups were similar to control which also indicates that these observations were not treatment-related.

Changes in postnatal survival:

not specified

External malformations:

not specified

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Ossification of the interparietal, occipital (supra-occipital), and sacral (neural) arch from females treated with 25 or 15 mg/kg bw/day A.I. Additionally, fetuses/litters from females receiving 25 mg/kg bw/day also showed a statistically significant reduction in incomplete ossification of parietal, jugal and hyoid regions. It is worth noting that reduced numbers of incomplete ossification of bones is also of no significance as treatment with the test items is normally associated with higher incidence of fetus with reduced ossification. Individual group mean values for the affected parameters were within or marginally below the historical control data ranges and these intergroup differences were considered to represent normal biological variation.

Visceral malformations:

not specified

Other effects:

not specified

Key result

Dose descriptor:

NOEL

Effect level:

25 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: No treatment-related changes were detected in the uterine parameters measured or on embryofetal development

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

25 mg/kg bw/day

Treatment related:

no

Relation to maternal toxicity:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

DISCUSSION

The administration of Quaternary ammonium compounds, C12-18-alkylbis (hydroxyethyl) methyl, chlorides (CAS No 71808-53-2) was well tolerated. Two females treated with 25 mg/kg bw/day A.I. showed clinical signs of noisy respiration on some days which may be due to an irritant nature of the test item. There was no effect of treatment with the test item at any level on maternal body weight development or associated food consumption. Additionally, no macroscopic findings were detected for any of the animals on the study whilst uterine and fetal examination also did not reveal any effect of the treatment on uterine parameters examined or fetal development.

A high dose level of 25 mg/kg bw/day A.I. was chosen taken into consideration the results from a previous OECD 422 study with this test item (Harlan Study Number: B61198) which showed treatment-related deaths at dose levels of 50 mg/kg bw/day or above. Additionally, animals treated with 25 mg/kg bw/day or above showed acute/subacute necrotizing inflammation and/or hyperkeratosis/acanthosis of the forestomach mucosa, which were considered to be to be a local effect consequent to an irritant nature of the test item administered as a bolus of gavage.

Conclusions:

The oral administration of Quaternary ammonium compounds, C12-18-alkylbis (hydroxyethyl)methyl, chlorides (CAS No 71808-53-2) to pregnant rats by oral gavage from gestation Days 3 to 19 at dose levels of 5, 15 and 25 mg/kg bw/day A.I. (incorporating a correction factor for 75% purity), resulted in no treatment related effects. The No Observed Effect Level (NOEL) for the pregnant female was considered to be 25 mg/kg bw/day A.I.

No treatment-related changes were detected in the uterine parameters measured or on embryofetal development. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 25 mg/kg bw/day A.I.

Executive summary:

The developmental toxicity / teratogenicity of the test substance, 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 414 (Prenatal Developmental Toxicity Study) / EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study).

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 the study no treatment-related changes were detected in the uterine parameters measured or on embryofetal development. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 25 mg/kg bw/day A.I.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available