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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
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
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.

Test animals

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.

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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.

Maternal developmental toxicity

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.).

Effect levels (maternal animals)

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

Results (fetuses)

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.

Effect levels (fetuses)

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

Overall developmental toxicity

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.