Dihexyl ether

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 April 2019 - 04 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This study was performed for the notification in other regions where authorities do not accept read-across data.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

d.d. 03 Aug 2018

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dihexyl ether

EC Number:

203-987-8

EC Name:

Dihexyl ether

Cas Number:

112-58-3

Molecular formula:

C12H26O

IUPAC Name:

1-(hexyloxy)hexane

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI Wistar rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: approx. 6 weeks
- Weight at study initiation: Males: 202–240 g, females: 152–186 g
- Fasting period before study: No
- Housing: Rodents were housed 2 or 3 animals of the same sex and group/cage. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities.
- Diet: Animals received ssniff® SM R/M-Z+H "Complete diet for rats and mice –breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum
- Water: tap water from the municipal supply ad libitum
- Acclimation period: 7/8 days (m/f)

DETAILS OF FOOD AND WATER QUALITY:
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided analytical certificate for the batch used, which is archived with the raw data.
Water quality control analysis was performed at least once every three months and microbiological assessment is performed monthly. The quality control results are retained with the raw data in the archives at the test facility. The water was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.3–25.1 (target: 22±3)
- Humidity (%): 32–72 (target: 30–70)
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 04 April 2019 To: 04 July 2019

During the study the relative humidity was out of the targeted range of 30-70%. The actual range was 32-72%, however as justified by the clinical veterinary it had no effect on the animals.
During the study the temperature of the room was out of the targeted range of 22±3°C. The actual range was 19.3-25.1°C, however as justified by the clinical veterinary it had no effect on the animals.

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The oral route was selected as it is the most relevant route of human exposure.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
The test item was incorporated into ssniff®, to generate the test concentrations required. Based on previous experimental work with the test item, the prepared diets contained approx. 20% less test item than the nominal concentrations (the added amount of test item mg per kg diet). Partial evaporation of the test item during the pelleting process was considered to be the reason for this. Therefore, the concentrations ordered from ssniff® had a correction factor of 1.25. Nominal levels were 1875 ppm, 6250 ppm and 18750 ppm for the low, mid and high dose groups which were expected to yield dietary concentrations of 1500 ppm, 5000 ppm and 15000 ppm in the low, mid and high dose groups.
The pelleting process was considered not to induce an "unmixing" of the diets or particle segregation, and would prevent the potential settling out of the fine-heavy particles that could occur in handling/transport of powder diets. Di-n-hexyl ether was incorporated into the diet and mixed for up to approximately 16 minutes (approximately 8 minutes for premix preparation, and approximately 8 minutes for preparation of the complete diets). Following mixing, pellets were prepared by simple compression; no binding agents, steam, external heat, any other process or substance were used that might affect the test item or the quality of the diets. Similar diet preparation procedures were used to generate control diet (0 mg Di-n-hexyl ether/kg diet).
The prepared diets were stored frozen (approx. -20°C) in plastic bags soon after preparation, pending and during transport to the testing facility, the prepared diets were also stored frozen. Each morning, an appropriate amount of frozen diet was thawed at room temperature, then placed into the animal cages. The next day, the remaining food was replaced with freshly thawed food in the animal cages. This way, the diet was at room temperature for not more than 27 hours during the study.
The stability of the test item in the diet had been verified for a period of at least 10 weeks frozen (approx. -20°C), and for 27 hours at room temperature after thawing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical sample measurement of the diets was performed from five different diet containers at each occasions from each concentration. Additionally, at two sample was similarly taken from the control diet, for analysis of Di-n-hexyl ether concentration (a total of 17 samples were measured per occasion).

Acceptable range for concentration analyses is 80% - 120% of the expected concenration.
Acceptance criteria of the homogeneity was that the CV of replicates (from 5 different locations) should be less than 10 RSD %.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 500 ppm

Remarks:

Average measured concentration 1440 ppm

Doses / concentrations 2

Dose / conc.:

5 000 ppm

Remarks:

Average measured concentration 4986 ppm

Doses / concentrations 3

Dose / conc.:

15 000 ppm

Remarks:

Average measured concentration 15517 ppm

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were set by the Sponsor in consultation with the Study Director, based on the available data and information from previous experimental work, including the results of a 28-Day Dose Range Finding Toxicity/Palatability Study in Rats performed at the Test Facility, where no significant toxicity was seen up to and including 15000 ppm (calculated dose intake ~1000 mg/kg bw/day) (See any other information on materials and methods).
- Fasting period before blood sampling for clinical biochemistry: Yes

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Principles and criteria summarized in the OECD Humane Endpoints Guidance Document No. 19 were taken into consideration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first treatment (on Day 0 male/female) and weekly thereafter, in the morning hours (am) and once before necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: weekly and prior to necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in all animals before treatment (Day -3/-4), and in the Control group and High dose group animals, during Week 13 (Day 87/88)
As no treatment related alterations were found, no additional animals of the Mid and Low dose groups were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day of necropsy
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (o/n)
- How many animals: all animals
- Parameters were examined acoording to the OECD TG 408 guideline.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of necropsy
- Animals fasted: Yes (o/n)
- How many animals: all animals
- Parameters were examined acoording to the OECD TG 408 guideline.

URINALYSIS: Yes
- Time schedule for collection of urine: over approximately 16 hours, overnight before necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters were examined acoording to the OECD TG 408 guideline.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 12 of treatment
- Dose groups that were examined:
- Battery of functions tested: FOB including sensory activity, landing foot splay grip strength and motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
After exsanguination the external appearance was examined, all orifices, and the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.
Organ weights were collected according to the OCED TG 408 guideline.

HISTOPATHOLOGY: Yes
On completion of the macroscopic examination tissues and organs were retained from all animals according to the OECD TG 408 guideline. Full histopathology was
performed in Groups 1 (Control) and 4 (High dose). In addition, any organs or tissues with macroscopic abnormalities (except minor changes) were subjected to histological
examination from all groups.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package of SAS 9.2 software package (within the validated Provantis system). The following decision tree was applied automatically for statistical evaluation of continuous numeric data.
The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis is the better option when the normality and heterogeneity assumptions implicit in the tests are adequate. If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach is not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
For non-continuous data, the Cochran-Armitage test for trend was applied and the Chisquared test was used for statistical differences relative to control.
For pathology data (macroscopic and microscopic data) the Cochran-Armitage test for trend was applied, then if appropriate, the Chi-squared test homogeneity test. If significance was plausible based on a user-defined value (0.05), a pairwise test of each treatment group versus the control group was made. If the group size was <5 then Fisher’s Exact Test was used, if the group sizes were bigger then the Chi-squared test was used; identifying differences of <0.05, <0.01 or <0.001 as appropriate.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No significant effect on male body weight or weight gain was seen in any treatment group. A test item-related effect was noted on body weight and/or body weight gain in the females of the High and Mid dose groups (15000 and 5000 ppm, respectively) when compared with controls. The effect on High dose was considered to be adverse in females, with a persistent and progressive difference, reaching almost 10% lower body weights than controls at termination; the difference for the Mid dose female group was small with no statistically significant effect at termination, hence this small difference was not considered to be adverse. There was no such effect observed in Low dose females (1500 ppm).
The overall body weight gain difference relative to control was statistically significant for High dose females when compared with controls (-19.2%). The overall body weight
gain difference for Mid dose females did not attain statistical significance but was 12.6% inferior to controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was a treatment-related effect on food consumption in females in the High dose group (15000 ppm) which was considered to be related to the slightly lower body weight. Low and Mid dose females and all treated males were unaffected. The lower food intake in High dose females may have been a palatability effect, there was no evidence of an adverse toxic effect. In males, there were no effect of the Test Item in any of the dose groups when compared to the control value.
There was a transient reduction in food consumption for the first two weeks of the treatment period in the High dose (15000 ppm) females, which is indicative of a palatability effect. However, it cannot be proven the reduced female High dose body weight and food intake were not adverse, hence the body weight in High dose females is conceded as adverse. Evaluation of the total food consumption over the treatment period showed a statistically significant reduction in food consumption for High dose females when compared with controls.

Food efficiency:

no effects observed

Description (incidence and severity):

The test item intake values (mg/kg bw/day) were calculated from the weekly individual body weight, weekly mean food intake and nominal diet concentration. The mean achieved dose levels (combined for males and females) were approximately 1234, 400 and 116 mg/kg bw/day in the High (15000 ppm), Mid (5000 ppm) and Low (1500 ppm) dose groups, respectively.
There were no effects of treatment on food conversion efficiency.

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were observed in the haematology parameters.
Statistically significant differences from controls were recorded for reduced PTT times in High dose males and females, and Mid dose males. These changes were considered to be incidental, there was no relationship with dose and all recorded values were normal and had minor difference to the Control. These reductions were considered to not reflect an adverse effect of the test item.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes in the clinical chemistry parameters that were considered to be related to treatment.
Statistically significantly differences were evident for the cholesterol and bile acid values of High dose females only when compared with controls. However, these changes were not seen in males, the cholesterol values were all within the historical control range and for bile acids, when the range of results in the concurrent control and lower dose groups are examined, the High dose values all fall within that range. In the absence of any associated histopathology changes, these numerical differences were not considered to represent an adverse effect.
In the female Mid dose group, the statistical difference for serum urea concentrations was considered incidental and not test item-related. No statistical significances were recorded
in the Low and High dose groups.
In High dose females the alkaline phosphatase (ALKP) was statistically significantly higher, however with higher standard deviation, cause by one individual value, other individual values were close to the historical control range and the Control values, therefore this was not considered related to the Test Item.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.
There was no adverse effect of treatment noted during the assessment of grip strength, foot splay or motor activity. Statistically significantly decreases in forelimb grip strengths were observed in the male dose groups. These were considered as incidental and not to reflect an effect of the test item; the differences did not show a clear dose response and all means
were well within the historical control range. No such differences were seen in the females.
All dose groups of males and females had a normal locomotor activity: in all cases, the initial activity was high, with a normal reduced mean activity in each 5-minute period to an approximate plateau by about 20-30 minutes. There was no statistical significance between the test item treated animals and the Control when evaluating the overall distance travelled (0-60 min, cm). The test item did not increase or decrease the normal locomotor activity.
In the landing foot splay test, for males in the High dose, values were statistically significantly lower than the Control, however these were well within the historical control range, therefore was considered to unrelated to the Test Item.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The male kidney weights (relative to body weight and brain weight) were statistically significantly higher than Control in the High Dose group (p<0.01 and p<0.05 respectively). The kidney weights were larger by 16.2%, related to body and by 15.5% related to brain weights.
The male liver weights were statistically significantly higher than Control in the High Dose group by 15.3%, related to body weight (p<0.01).
The male thyroid/parathyroid weights were statistically significantly higher than Control in the High Dose group by 16.1%, related to body weight (p<0.05).
The absolute and relative to brain weights of the testes were statistically significantly higher than Control in the Low Dose group the absolute by 9.9% and the related to body weight by 10.7% respectively, without a dose response relationship.
Terminal body weights of High Dose females were statistically significantly lower by 9.4% compared to controls.
The relative (to body) weight of the brain was statistically significantly higher than Control in the High dose female group by 12.4%, but the absolute weights were not statistically different.
The absolute and relative to brain weights of the adrenals were statistically significantly lower than Control in High dose female group the absolute by 13.9% and the related to brain by 15.9% respectively; but not statistically different relative to body weight.
The kidney weights were statistically significantly higher than Control in the High dose female group by 15.4%, related to body weight (p<0.01).
The liver weights (absolute and relative to body and brain) were statistically significantly higher than Control in the High females (p<0.01). The absolute liver weights were larger by 17.5%, by 29.5% related to body weight and by 15.0% related to brain weights.

There was no clear histopathological correlate for the weight changes neither in male or female High Dose animals for any of the above organs. The statistical differences in female brain and adrenals were considered to be due to smaller terminal bodyweights at termination, and not a direct test item effect on the organs. The male relative (to body weight) thyroid weight was above the concurrent control mean value, but in the historic mean value, so was not considered to be an effect of the test item.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All of the animals survived the treatment period and no test item-related changes were observed at necropsy.
The recorded findings, were focal dark red discoloration of the adrenals (in 1/10 Low dose female), the brown raised area in the liver (in 1/10 High dose male and 1/10 Low dose female), enlarged kidney (in 1/10 High dose male), multifocal dark red discoloration of the glandular mucosa (in 1/10 High dose male) and raised area of the non-glandular region (1/10 Control male) of the stomach, enlarged testis (in 1/10 High dose male), small testes, prostate and epididymis (in 1/10 Mid dose male), the enlarged (in 1/10 Mid dose male) or dark red (in 1/10 Control male) thymus, cyst in the pituitary (in 1/10 High dose male), enlarged mandibular lymph node (in 1/10 High dose and 1/10 Low dose males) and dilatation and discoloration of the uterine cervix/body/horn (through Control and dosed female groups). Based on the low severity and incidence of these findings through control and dosed groups, all were considered as incidental or background.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No adverse test item-related findings were observed in any group.
The recorded changes were increased vacuolation (1/10 Control and 1/10 High dose males) and diffuse congestion (in one Low dose female) in the adrenals and centrilobular, hepatocellular vacuolation (in 2/10 High dose male, 1/10 Control male and 1/10 Control female) in the liver, focal/multifocal degeneration of cardiomyocyte (in 2/10 Control and 1/10 High dose males and 1/10 Control female), tubular basophylia (in 1/10 High dose male), hyaline casts (in 3/10 Control males and 1/10 Control female), mineralization (in 1/10 High dose female) and pyelonephritis (in 1/10 High dose female), Harderian metaplasia in the lacrimal gland (in 2/10 Control and 3/10 High dose males), increased cellularity in the cortex of the mandibular lymph node (in one Low dose and in 1/10 High dose males), focal congestion/haemorrhage in the gastric mucosa (in 1/10 High dose male), degeneration/atrophy, or dilatation tubule in the testis (in 1/10 Control, one Mid dose and in 2/10 High dose males), cell debris in the epididymis (in one Mid dose male), inflammatory cell infiltrate in the prostate (in 2/10 Control males), congestion/haemorrhages in the thymus (in 3/10 Control males), and signs of oestrus in the uterus. The histological findings seen in treated animals were generally also seen in concurrent control, or were without meaningful differences in severity and incidence, therefore were regarded as incidental or a common background changes.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Absence of adverse effects up to and including the highest dose level (15517 ppm).

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

DRF

The dose selection of this study was based on available toxicity data provided by the Sponsor. Accordingly, three groups of eight Wistar rats (4/sex/group) were fed with diet containing Di-n-hexyl ether at 1500, 5000 and 15000 ppm (mg/kg diet) for 28 consecutive days (nominal concentrations). The control group received basal diet. The concentrations of Di-n-hexyl ether in the diets were determined using a validated GC-FID method.

Clinical and mortality observations were performed twice daily. Body weight and food consumption were measured regularly. Following the daily repeated dose administration for 28 days, blood samples were collected for clinical pathology at necropsy from the animals. Gross macroscopic examination was performed at necropsy in each animal. Selected organs were weighed, and selected tissues were preserved in fixative.

Results

Di-n-hexyl ether administered in diet at nominal concentrations of 1500, 5000 and 15000 ppm, was equivalent to dietary doses of approximately 104, 346 and 974 mg/kg bw/day in the Low, Mid and High dose groups, respectively, taking into account the analytical results (~80% of nominal concentrations).

No test item-related clinical signs, adverse body weights or food consumptions were noted during the study and no macroscopic lesions were seen at necropsy.

Higher liver weights were recorded in the male and female High dose groups and higher kidney weights in the female Mid and High dose groups. These were considered to be probably non-specific adaptive changes to test item in the liver and kidney.

There were no treatment-related statistically significant differences among treated groups at any dose levels in the haematology or clinical chemistry parameters.

In conclusion, no adverse effects were identified in rats given up to and including 15000 ppm Di-n-hexyl ether (nominally) in diet.

Based on the analytical results, it is suggested that for the next 90-day study the diets should be prepared with adding 125% of the nominal concentrations, to allow for potential evaporation during the pelleting process. Using a factor of approximately 3, the nominal dose levels suggested are 1875, 6250 and 18750 ppm, which are expected to be approximately 100, 300 and 1000 mg/kg bw/day actual dose levels.

Dose concentration and test item intake:

Group number	Group designation	Nominal concentration in diet (ppm)	Target concentration in diet (ppm)	Target dose levels (mg/kg bw/day)	Average measured concentrations	Mean test item intake Males (mg/kg bw/day)	Mean test item intake Females (mg/kg bw/day)	Mean test item intake combined (M+F) (mg/kg bw/day)
1	Control	0	0	0	0	-	-	-
2	Low	1875	1500	100	1440	108.8	122.9	115.9
3	Mid	6250	5000	300	4986	373.3	426.6	400.0
4	High	18750	15000	1000	15517	1163.8	1303.7	1233.8

Results analytical verification of concentrations:

The mean Di-n-hexyl ether concentrations evaluated for the test item containing diet samples were in the range of 90-112% of the expected concentration. These results were within the acceptable range (80%-120% of the expected concentration) and were considered to be suitable for the study purposes.

All formulations proved to be homogeneous. The relative standard deviation of the replicates was 1.1 -4.0%.

Applicant's summary and conclusion

Conclusions:

In a 90-day repeated dose toxicity study according to OECD TG 408 and in accordan with GLP principles the NOAEL for the test item is considered to be 400 mg/kg bw/day for females and ≥1000 mg/kg bw/day for males.

Executive summary:

The oral repeated dose toxicity of dihexyl ether was tested by administration of the substance to Wistar rats for 90 consecutive days according to OECD Guideline No. 408.

Ten male and ten female Wistar rats/group were fed by diet containing di-n-hexyl ether at 3 concentrations (ppm) for 90 consecutive days ad libitum. The control group received control diet ad libitum. Before the onset of the 90-day treatment period, all animals received the control diet for 7 days. The achieved dose of test item in diet was a mean of approximately 1440, 4986 and 15517 ppm, with a test item intake of approximately 116, 400 and 1234 mg/kg bw/day for the low, mid and high dose groups respectively.

There was no mortality during the study and no test item-related clinical signs were observed during the study. The only adverse effects on the body weights were slight reductions in High dose females. Other dose groups had little or no difference compared with controls. There were no adverse effects on food consumption or neurological assessment in any of the dose groups.

At clinical pathology, no adverse test item-related changes were seen. No macroscopic changes were seen at necropsy. Liver and kidney weights were higher than control in High dose males and females, but the change was considered to be adaptive and non-adverse.

There was no adverse test item related histopathology findings.

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for the test item is considered to be 400 mg/kg bw/day (Mid dose) for females and at ≥1000 mg/kg bw/day (High dose) for males.