Quaternary ammonium compounds, C12-14-alkyl(hydroxyethyl)dimethyl, chlorides

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 December 2009 to 07 February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

[see concordance under 1.1. of the CSR]

IUPAC Name:

[see concordance under 1.1. of the CSR]

Details on test material:

Batch No.: DEG4052477
Chemical Name: Quaternary ammonium compounds,
C12-18-alkyl(hydroxyethyl)dimethyl, chlorides
Density: 1.00 g/cm3
Active Componetns (%): 98.8 % (COA dated 10.06.2009)
Unidentified Components: Free Amine 0.4 %
Hydrochloride 0.2 %
Water (KF) 1.1 %
Colour: Colourless
pH: 8.0 DIN EN 1262
Physical State: Solid
Purity: 98.8 %
Expiry Date: 01 March 2011
Storage: Room Temperature (RT)
Stability in Solution/Suspension: Stable
Melting Point: 195ºC

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

-strain: Wistar Crl:WI
-Source: Charles River, 97633 Sulzfeld, Germany
-The animals were derived from a controlled full barrier maintained breeding system (SPF)
-Age of the animals at the beginning of the study was 8-9 weeks. The range of the body weight was:
Females: 175 to 203 g, (mean: 184.95 g, ± 20%= 36.99 g)
Males: 257 to 290 g, (mean: 272.63 g, ± 20%= 54.53 g)
-Housing-The animals were kept individually in IVC cages, type III H, polysul-phone cages on Altromin saw fibre bedding
-Diet (ad libitum): Altromin 1324 maintenance diet for rats and mice
-Water (ad libitum): tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, muncipal residue control, microbiol. controlled periodically)
-Acclimation period 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 3 °C
- Relative humidity: 55 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour

IN-LIFE DATES: from 15 December 2009 to 07 February 2010

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
-Amount of vehicle: 10 mL/kg bw
-Concentration in vehicle: 1.5, 3.0, 6 mg/mL

Details on mating procedure:

mating ration 1:1, mating period- 14 days

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration was analyzed for nominal concentration. Homogeneity of the test item in the vehicle was analyzed for the low and high dose concentrations.
Samples for the nominal concentration verification were taken in study week 1
(First week of pre mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation).
Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation in study week 1 and 5.
The dose formulation analysis was performed at BSL BIOSERVICE Scientific Laboratories GmbH.
The analytical report (BSL Study 093602) is added in Annexure 4.

Duration of treatment / exposure:

males: 28-29 days; females: Approx 54 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 15, 30, 60 mg/kg body weight
Basis:
nominal in water

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The animals were dosed with the test item on 7 days per week basis. The test substance was administered daily during 14 days pre mating and 14 days mating in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28-29 days.
The test item was administered by gavage using a gavaging cannula. The maximum dose volume administered was 10 mL / kg body weight.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

Cage Side Observations:
-Time Schedule: Along with clinical observations
-Cage side observation included: spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnea, asphyxia, vocalization, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), and piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour

Detailed Clinical Observations:
-Time schedule: General Clinical observation were made twice a day

FOB:Sensory reactivity to different modalities, grip strength and motor activity assessments and other behaviour observations were conducted on five randomly selected males and females from each group. Observation was made during the last week of treatment in males and on day 3 of the lactation in females (only lactating females were evaluated).

Body weight:

-weighed at randomisation; males weekly during the entire study period and at terminal sacrifice; females were weighed weekly during pre mating period, on gestation day 0, 7, 14, 20 and on PND 1 (within 24 hours of parturition) and 4 along with pups.

Food consumption:

-measured on corresponding day of body weight (in males only during pre mating period) after beginning of the dose administration. Food consumption was not measured during mating period.

Litter Observations:
-The duration of gestation was recorded and is calculated from day 0 of pregnancy.
-Each litter examined soon after delivery of the dam for the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities
-Live pups counted, sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum

Hematology:

-Time schedule for the collection of blood: day of necropsy
-Anaesthetic used for the blood collection: yes
-Animals fasted- no
-Parameters: Haematocrit, haemoglobin, erythrocyte count, total and differential leucocyte count,
platelet count, blood clotting time , and differential leucocytes count (On five randomly selected males and females)

Clinical Biochemistry:

-Time schedule for collection of blood: day of necropsy
-animals fasted: no
-Parameters: sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein and albumin, two enzymes indicative of hepatocellular effects ( alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase)- on five randomly selected males and females.

Urinalysis:

-Time Schedule for the collection of urine: day of necropsy- samples collected from five randomly selected males at the terminal sacrifice.

Sacrifice and pathology

Gross Pathology-
-Males were sacrificed after the completion of mating period (total dosing of 28-29 days)
-females were sacrificed on respective post natal day 4 along with pups by using high dose of sodium pentobarbital.
-In the animals randomly selected for the blood collection, anaesthesia solution (Ketamin/Xylazin, 2:1) was used. At the time of sacrifice or death during the study, the adult animals were subjected to a full, detailed gross necropsy which includeed careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents with special attention was paid to the organs of the reproductive system.
-The number of implantation sites and corpora lutea recorded for each parental female at necropsy
-Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter were carefully examined for gross abnormalities
-The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicle with coagulating glands as a whole), and all organs showing macroscopic lesions of all adult animals were preserved
-Organ weight: Reproductive organs from all animals were weighed (testes, epididymides, prostate, seminal vesicle with coagulating glands as whole, ovaries, uterus with cervix as applicable). From five adult males and females randomly selected from each group, the wet weight of the liver, kidneys, adrenals, thymus, spleen, brain and heart were taken.

Histopathology:

Full histopathology was carried on the preserved organs and tissues of same five randomly selected animals in control and high dose group. Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) and all organs showing gross lesions were examined in all animals. In addition, the evaluation of stomach (non glandular and glandular) and trachea were extended for LD and MD groups due to findings in HD group animals.
The organ list were as follows;
- all gross lesions
- brain (representative regions including cerebrum, cerebellum and pons)
- spinal cord
- mammary glands (female only)
- liver
- kidneys
- adrenals
- stomach
- small and large intestines (including Peyer´s patches)
- thymus
- thyroid
- spleen
- trachea and lungs
- heart
- urinary bladder
- lymph nodes (one lymph node covering the route of administration (mesenteric) and another one distant from the route of administration to cover systemic effects (axillar)
- peripheral nerve (e.g. N. ischiadicus/ N. tibialis) in close proximity to muscle
- section of bone marrow (sternum)


Histopathological processing was performed at Propath UK Ltd, Willow Court, Netherwood Road, GB - Hereford HR2 6JU.
Histopathological evaluation performed at GLP-certified test site KALEIDIS –Consultancy in Histopathology, 6 rue du Gers, 68300 Saint-Louis, France.

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

-Each litter to establish the number of pups, sexof pups, stillbirths, live births, runts and for the gross abnormalities.
-Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum.
- In addition to the observations on parent animals abnormal behaviour of the offspring, if any, was recorded.

Postmortem examinations (parental animals):

-hematology on blood samples
-clinical biochemistry
-urinalysis
-Macroscopic examination
-organ weight
-histopathology

Postmortem examinations (offspring):

-gross external examination
-full necropsy of abnormal/dead pups

Statistics:

For statistical analysis one-way analysis of variance (ANOVA) followed by Dunnett‟s multiple comparison test was carried out to reveal any differences between control- and test groups. These statistics were performed with GraphPad Prism V.5 software (p<0.05 was considered as statistical significant).

Reproductive indices:

copulation index, fertility index, delivery index and viability index were calculated based on the data generated

Offspring viability indices:

between PND 0 and PND 4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The clinical signs observed in female control and treatment groups are as follows.
Control- nibbling of fur;
LD, MD and HD- respiratory sound, salivation;
MD and HD- moving the bedding, piloerection, vaginal secretion, nibbling of fur, regurgitation, sneezing;
HD- weight loss, dyspnoea, cyanosis, coughing.
The incidences of clinical signs observed were higher in MD and HD groups of both sexes and could be attributed to toxicity.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item related mortalities and effect on functional and behavioural endpoints were observed in male and female animals during the entire study period. Three animals (1 female and two males) died prematurely in the study, which was due to gavaging error.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant difference was observed for body weight development in male HD group during premating days 1-7 and premating day 1 to terminal sacrifice [1- TS (day 29/30)] and female MD and HD group during GD 14-20 compared to corresponding controls. The statistical deviation observed in Male HD group and female MD and HD groups may be attributable to the observed reduced food intake during the same period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the high dose of 60 mg/kg bw/day as well as in one dam of the mid dose with 30 mg/kg bw/day local irritating effects at the forestomach were observed. These findings are considered a direct consequence of the corrosive property of the test substance. Findings in the trachea are considered to be secondary effects due to reflux (regurgitation) and subsequent aspiration of gavaged test substance with following repetitive direct irritation of the nonglandular mucosa of the stomach by the test substance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY

All animals survived except one female from the high-dose group (Animal no. 35) and two males from the high-dose group (Animal no. 73 and 79) till the end of the study.

No clinical signs were observed in control and LD groups of males. The following clinical signs were observed in MD and HD group animals.
MD- salivation, moving the bedding post application, piloerection, respiratory sound, alopecia; HD- salivation, moving the bedding post application, piloerection, respiratory sound, half eyelid closure, regurgitation, bloody nose. The clinical signs observed in female control and treatment groups are as follows. Control- nibbling of fur; LD- respiratory sound, salivation; MD- respiratory sound, salivation, moving the bedding, piloerection, vaginal secretion, nibbling of fur, regurgitation, sneezing; HD- respiratory sound, salivation, moving the bedding, piloerection, weight loss, dyspnoea, cyanosis, coughing. The incidences of clinical signs observed were higher in MD and HD groups of both sexes and could be attributed to toxicity.
Three animals (1 female and two males) died prematurely in the study, which was due to gavaging error.

NEUROBEHAVIOUR

No relevant differences were observed in males and females.

HEMATOLOGY
Except for RBC (male HD group), MCHC (Female MD group), PTT and aPTT (female LD group) no statistical significant deviation was recorded in any of the hematological parameters between the treatment group and the corresponding control values. The statistical deviation observed for MCHC (Female MD) and RBC (Male HD) may not be attributed to toxicity due to lack of dose response effect.


CLINICAL BIOCHEMISTRY

Except for SGPT values in male HD group no deviation was recorded in any of the parameters. This deviation recorded in SGPT value could be due individual incidental value and may not be attributed to the treatment as no microscopic findings were recorded in liver that could support this finding.

URINALYSIS

no effects

ORGAN WEIGHT

In males, except for absolute seminal vesicle weight in HD group, statistically no significant differences in the absolute and relative organ weights of the treatment groups was observed when compared with the controls. The deviation observed in absolute seminal vescicle weight cannot be attributed to toxicity as there was no such deviation recorded for relative seminal vesicle weight.
In females, statistically no significant differences in the absolute and relative organ weights of the treatment groups was observed when compared with the controls.

GROSS PATHOLOGY

At terminal sacrifice, macroscopic organ findings noted were mainly in MD nad HD group animals.

In males, the macroscopic findings observed were multiple white spots on mucosa of cardis (animals 66, 72, 74, 75 and 78); lung filled with foamy and bloody content (animals 73 and 79); white and thickening at mucosa cardia (animal 76, 80).
In females, macroscopic findings observed were discoloured lung with multiple spots (animals 21 and 27); multiple white/yellowish/greenish spots on mucosa of cardia (animals 31, 32, 33, 34, 37, 39 and 40); lung ,trachea, mouth and nose with bloody foam (animal 31) .

HISTOPATHOLOGY

In the female and male reproductive organs, no macroscopic or histopathological lesions considered to be test item-related were observed.

In the nonglandular part of the stomach, test item-related histopathological changes were noted in all animals evaluated from the HD group and in single animals evaluated from the MD group and comprised a multifocal/diffuse epithelial hyperplasia, mostly in combination with epithelial hyperkeratosis and, in a proportion of animals, with submucosal inflammatory edema.

In the trachea, a minor epithelial alteration/hyperplasia was seen in a small proportion of animals of the MD and HD groups.

REPRODUCTIVE PARAMETER

No significant difference was observed on precoital interval when compared with their corresponding controls. All pregnancies resulted in normal births. Successful mating resulted in 10/10 pregnancies in the control and LD groups and 9/10 pregnancies in MD and 8/9 in HD groups.

Slightly but not significant reduced fertility index (No. of pregnant females/No. of copulated females X 100) was observed in HD (88.9 %) and MD groups (90%) as compared to Control (100%) and LD groups (100%).

No significant differences observed for any of the reproductive indices (copulation index, fertility index and delivery index and viability index) between the treatment and control groups.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive toxicity, no major toxicological findings

Remarks on result:

other:

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

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

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

PRE and POST NATAL DATA

Group mean number of corpora lutea, number of implantation sites, number of live pups born on PND 0, percent pre implantation loss and post implantation loss remained unaffected due to treatment when compared with controls.

LITTER DATA

No treatment related effect was observed on total number of pups born, number of males, number of females, sex ratio, live pups, still birth and runt on PND 0 and total number of live pups and sex ratio on PND 4.

PUP SURVIVAL

Survival of the pups from PND 0 to PND 4 remained unaffected due to treatment in all treatment groups.
At necropsy, no gross external abnormalities considered to be treatment related.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

gross pathology

other: Number of live pups born on PND 0, number of males and females, sex ratio uneffected compared to controls. Survival of pups from PND 0 to PND 4 remained unaffected in all treatment groups. No gross pathological findings observed.

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, in this combined repeated dose toxicity and reproduction/ developmental toxicity screening test with HYEQS, the repeated dose administration of HYEQS in deionised water to the male (28-29 days) and female (maximum 54 days) Wistar rats at dosages of 15, 30 and 60 mg/kg body weight revealed no major toxicological findings.

Parameters like neurology, hematology, clinical biochemistry, organ weight and fertility in males and females remained unaffected. The deviation observed for body weight development and food consumption were not attributed to treatment. The prenatal and litter data also did not show any treatment related effect. However, the clinical/macroscopic findings observed in males/ females of MD and HD groups correlated with the microscopic findings (e.g. multifocal/diffuse epithelial hyperplasia in the nonglandular part of the stomach, mostly in combination with epithelial hyperkeratosis and with submucosal inflammatory edema and a minor epithelial alteration/hyperplasia in the trachea as noted in some animals). These findings are based on local effects due to the corrosive properties of the test substance.
Based on the data generated, the dosage 15 mg/kg/day is determined as No-Observed- Adverse-Effect-Level (NOAEL) for overall toxicity due to local histopathological findings in stomach and trachea of the parental animals. However, the No Observed Adverse Effect Level (NOAEL) for reproductive toxicity is believed to be 60 mg/kg /day in males and females

Executive summary:

Material and Methods

In this Combined Repeated Dose Oral Toxicity Study with the Reproduction/Developmental Toxicity Screening Test performed according to First Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 422, “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” adopted on 22 March 1996 in wistar rats with HYEQS suspended in deionised water as vehicle, orally administered in graduated doses to three groups of male and female rats (Wistar Crl:WI) by gavage using feeding tube. The main study included 4 groups (C, LD, MD and HD) with each 10 males and 10 females

The animals were dosed with the test item on 7 days per week basis. The test substance was administered daily during 14 days pre mating and 14 days mating in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28-29 days.

The test item was administered by gavage using a gavaging cannula. The maximum dose volume administered was 10 mL / kg body weight.

The following doses were chosen,

Low Dose: 15 mg/kg body weight

Mid Dose: 30 mg/kg body weight

High Dose: 60 mg/kg body weight

RESULTS

All animals survived except one female (Animal no. 35) and two males (Animal no. 73 and 79) till the end of the study. Male animals were sacrificed on day 29 and 30. Non pregnant females were sacrificed on the respective day 26 after the sperm positive vaginal smear as an evidence of mating. Lactating females along with pups were sacrificed on respective post natal day 4.

No test item related mortalities and effect on functional and behavioural endpoints were observed in male and female animals during the entire study period.

Statistically significant difference was observed for body weight development in male HD group during premating days 1-7 and premating day 1 to terminal sacrifice [1- TS (day 29/30)] and female MD and HD group during GD 14-20 compared to corresponding controls.

Statistically significant difference was observed for food consumption in male HD group during premating days 1-7.

Statistical analysis of reproduction and litter data revealed no treatment related effect on group mean litter weight, number of males, number of females, total litter weight, female litter weights on PND 0 and PND 4, percent pre implantation loss, post implantation loss, total number of pups born, sex ratio, live pups, still birth and runt on PND 0, total No. of live pups and sex ratio on PND 0 and 4, precoital interval, No. of corpora lutea, No. of implantation sites when compared with controls.

Statistical significant difference was observed for male litter weight on PND 0 and 4 in LD group, which had no toxicological relevance (as there was no dose dependence response recorded).

Survival of the pups from PND 0 to PND 4 remained unaffected due to treatment in all treatment groups.

In male and female, statistical significant effect was observed for RBC (male HD), MCHC (female MD), PTT (female LD) and aPTT (female LD) compared to their corresponding control, which had no toxicological relevance.

Urinalysis in five randomly selected males from each group revealed no test item related effect.

Statisitcal analysis of clinical biochemistry data revealed increase in SGPT value in male HD group compared to control. This finding may be attributed to individual incidental value.

Treatment related gross pathological findings were observed in the nonglandular part of the stomach in all animals evaluated from the HD group and in single animals evaluated from the MD group. The histopathological changes comprised of multifocal/diffuse epithelial hyperplasia, mostly in combination with epithelial hyperkeratosis and, in a proportion of animals, with submucosal inflammatory edema.

No test item related changes were observed in pups at necropsy or death during the study.

In males, statistically no significant differences in the absolute (except absolute seminal vesicle weight in male HD group) and relative organ weights of the treatment groups was observed when compared with the controls. The deviation of seminal vescicle weight was regarded as incidental.

The histopathological evaluation of male and female reproductive organs did not reveal any histopathological lesions considered to be treatment related.

CONCLUSION

In conclusion, in this combined repeated dose toxicity and reproduction/developmental toxicity screeing test with HYEQS, the repeated dose administration of HYEQS in deionised water to the male (28 -29 days) and female (Maximum 54 days) Wistar rats at dosages of 15, 30 and 60 mg/kg body weight weight revealed no major toxicological findings.

Parameters like neurology, hematology, clinical biochemistry, organ weight and fertility in males and females remained unaffected. The deviation observed for body weight development and food consumption were not attributed to treatment. The prenatal and litter data also did not show any treatment related effect.

However, the clinical/macroscopic findings observed in males/females of MD and HD groups correlated with the microscopic findings (e.g. multifocal/diffuse epithelial hyperplasia in the nonglandular part of the stomach, mostly in combination with epithelial hyperkeratosis and with submucosal inflammatory edema and a minor epithelial alteration/hyperplasia in the trachea as noted in some animals). These findings are based on local effects due to the corrosive properties of the test substance.

Based on the data generated, the dosage 15 mg/kg/day is determined as No-Observed_Adverse-Effect-Level (NOAEL) for overall toxicity due to local histopathological findings in stomach and trachea of the parental animals. However, the No Observed Adverse Effect Level (NOAEL) for reproductive toxicity is believed to be 60 mg/kg/day in males and females.