Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

There is data on developmental/reproduction toxicity on C12-18-alkylbis(hydroxyethyl)methyl, chloride, (CAS no 71808-53-2) from the available combinedrepeated dose toxicity study with the reproduction/developmental toxicity screening study (OECD 422). The overall reproductive performance of rats appeared to be unaffected by the administration of the test substance. Mating performance and fertility, corpora lutea count, duration of gestation, post implantation loss, litter size, and post natal loss were all unaffected by the test substance and any difference was within the range of the historical reference data.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
OEDC 422
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-02-21 to 2009-11-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3650
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat, HanRcc: WIST(SPF)
Sex:
male/female
Details on test animals 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)

IN-LIFE DATES: From: To:
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
Details on study schedule:
see table 1. - study schedule.
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.
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.
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.
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
Description (incidence and severity):
No effects on day 1 and day 4 post partum
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.
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.
Reproductive effects observed:
no
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
no
Relevant for humans:
no

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  -  -  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:

At 50 mg/kg bw/day, the evaluation of the reproduction/development parameters were carried out only on 5 females and it did not give an indication of any test substance related effect. Due to the number of females available for assessing possible reproductive/development effects, lower than the number required by the relevant guidelines (5 versus 8), it was not possible to definitively set the NOEL for reproductive and developmental toxicity at this dose level.

The overall indication from the study is that the test item will not affect reproduction or fertility in rats and therefore no classification is required.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study is performed according to OECD 422 guideline and under GLP and has reliability rating 1. It is sufficient to cover the information requirements in Annex VIII.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There is data on developmental/reproduction toxicity on C12-18-alkylbis(hydroxyethyl)methyl, chloride, (CAS no 71808-53-2) from the available combinedrepeated dose toxicity study with the reproduction/developmental toxicity screening study (OECD 422).The overall reproductive performance of rats appeared to be unaffected by the administration of the test substance. Mating performance and fertility, corpora lutea count, duration of gestation, post implantation loss, litter size, and post natal loss were all unaffected by the test substance and any difference was within the range of the historical reference data.

 

Short description of key information:

At 50 mg/kg bw/day, the evaluation of the reproduction/development parameters were carried out only on 5 females and it did not give an indication of any test substance related effect. Due to the low number of females available for assessing possible reproductive/development effects; 5 versus the required 8 in the relevant guideline, it was not possible to definitively set the NOAEL for reproductive and developmental toxicity at this dose level.

The overall indication from the study is that the test item will not affect reproduction or fertility in rats and therefore no classification is required.

Justification for selection of Effect on fertility via oral route:

An OECD 414 Pre-natal developmental test is available on C12-18-alkylbis(hydroxyethyl)methyl, chloride (CAS no 71808-53-2). The test has validity rating 1 and it is performed according to GLP and under current standards.

Justification for selection of Effect on fertility via inhalation route:

C12-18-alkylbis(hydroxyethyl)methyl, chloride (CAS no 71808-53-2) is handled as a liquid with a vapour pressure of 0.00073 Pa at 20°C and the identified uses are not expected to cause formation of aerosols, particles or droplets of inhalable size. Therefore it is not considered to be scientifically valid to conduct an inhalation study. Also the corrosive nature of the substance would make it difficult to perform such a test for animal welfare reasons.

Justification for selection of Effect on fertility via dermal route:

Due to the fact that C12-18-alkylbis(hydroxyethyl)methyl, chloride (CAS no 71808-53-2) is considered to be corrosive to skin, it is not possible to conduct repeat dose dermal toxicity studies due to animal welfare considerations. Also it is considered very unlikely that dermal absorption would exceed oral absorption. It is therefore expected that the oral NOAEL would be lower than the systemic one from a dermal study. Data from the repeat dose oral studies can be used in the setting of DNELs, following the ECHA guidance document for deriving DNEL values. As appropriate DNEL values can be calculated using the oral dosing study data, it is not justified on animal welfare grounds to perform a repeat dose dermal toxicity study.

Effects on developmental toxicity

Description of key information

The available study onC12 -18 -alkylbis(hydroxyethyl)methyl, chloride (CAS no 71808 -53 -2) is an OECD 414 Pre-natal developmental test, which showed no indications of reproduction/developmental effects. 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 effects were detected in the uterine parameters examined, in fetal viability or in growth and development. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 25 mg/kg bw/day A.I.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 09 July 2015 and 25 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
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 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
Dose / conc.:
15 mg/kg bw/day
Dose / conc.:
25 mg/kg bw/day
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.
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.
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.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic abnormalities were detected for any of the females.
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.
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
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.
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.
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:

Introduction

The study was performed according to the study plan and was designed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis. The study was designed to comply with the following guidelines:

  • US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)
  • Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)
  • OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)
  • Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Methods

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.

Resuls

Mortality

There were no unscheduled deaths.

Clinical Observations No clinical signs of systemic toxicity were detected at any dose level. Two females treated with 25 mg/kg bw/day showed instances of noisy respiration which were deemed to be due to an irritant nature of the test item.

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

Water Consumption There was no adverse effect on water consumption at any level.

Post Mortem Studies No macroscopic abnormalities were detected in females from any dose group.

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

Fetal Examination No treatment-related effects were detected on fetal external findings. No treatment-related effects were detected in the type and incidence of skeletal or visceral findings in fetuses from females treated with 5, 15 or 25 mg/kg bw/day A.I.

Conclusion

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The available study onC12 -18 -alkylbis(hydroxyethyl)methyl, chloride (CAS no 71808 -53 -2) is an OECD 414 Pre-natal developmental test, which showed no indications of reproduction/developmental effects. The substance is produced in volumes between 100-1000 tonnes per year, which requires an OECD 414 prenatal developmental study as required in Annex IX.

Short description of key information:

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.

Justification for selection of Effect on fertility via oral route:

AnOECD 414 Pre-natal developmental test is available on C12-18-alkylbis(hydroxyethyl)methyl, chloride (CAS no 71808-53-2).

The test has validity rating 1 and it is performed according to GLP and under current standards.

Justification for selection of Effect on fertility via inhalation route:

C12-18-alkylbis(hydroxyethyl)methyl, chloride (CAS no 71808-53-2) is handled as a liquid with a vapour pressure of 0.00073 Pa at 20°C and the identified uses are not expected to cause formation of aerosols, particles or droplets of inhalable size. Therefore it is not considered to be scientifically valid to conduct an inhalation study. Also the corrosive nature of the substance would make it difficult to perform such a test for animal welfare reasons.

Justification for selection of Effect on fertility via dermal route:

Due to the fact that C12-18-alkylbis(hydroxyethyl)methyl, chloride (CAS no 71808-53-2) is considered to be corrosive to skin, it is not possible to conduct repeat dose dermal toxicity studies due to animal welfare considerations. Also it is considered very unlikely that dermal absorption would exceed oral absorption. It is therefore expected that the oral NOAEL would be lower than the systemic one from a dermal study. Data from the repeat dose oral studies can be used in the setting of DNELs, following the ECHA guidance document for deriving DNEL values. As appropriate DNEL values can be calculated using the oral dosing study data, it is not justified on animal welfare grounds to perform a repeat dose dermal toxicity study.

Justification for classification or non-classification