Hexanamide, N-(2-hydroxyethyl)-3,5,5-trimethyl-

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

The vapour pressure of the test substance was determined using gas saturation apparatus according to EU Method A.4 (Wicks, 2013).

Vapour pressure:

0 Pa

at the temperature of:

25 °C

.

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Sep 2012 to 30 Jan 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant with current guidelines and GLP compliant.

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

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 8-9 weeks
- Weight at study initiation: Males: 303-372g: Females: 215-265g
- Fasting period before study: None
- Housing: 2 or 3 per sex per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23C
- Humidity (%): 40-70%
- Air changes (per hr): Minimum of 10
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 24 Sep 2012 To: 16 Nov 2013

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item is miscible with corn oil but not with water or other vehicles.
- Concentration in vehicle: 10-100 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): Not applicable
- Purity: Not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Achieved concentration and homogeneity determined to be within acceptance criteria at Weeks 1 and 4 of study.

Duration of treatment / exposure:

Once daily oral gavage adminsitration as follows:
Males: 28 days
Females: between 42 and 46 days (dependent upon dates of mating and parturition)

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
Doses: 0, 30, 75 and 125 mg/kg/day
Basis:

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:The dose levels were agreed with the Sponsor following evaluation of existing relevant toxicological data, including a 7-day dose range finding study (Charles River Study No. 496177).
- Rationale for animal assignment (if not random): Not applicable- random allocation
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
- Section schedule rationale (if not random): Not applicable

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals checked hourly up to 4 hours post dose on each day of dosing
- Cage side observations: Twice daily mortaility checks (am/pm)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Once during pre-trial and then daily throughout the treatment period.

FOOD CONSUMPTION
- Food consumption: Weekly prior to pairing for mating. No further food consumption for the males, however food consumption measured for females on Days 0-7, 7-14 and 14-20 of gestation and 0-4 of lactation.

WATER CONSUMPTION Yes
- Time schedule for examinations: Water consumption was monitored regularly by visual inspection of water bottles however no data was recorded.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-trial, Week 4 (males) and prior to sacrifice (females in early lactation).
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 4 (males), ~ Day 4 of lactation (females)
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 5 animals per sex per dose group
- Parameters examined:

Red blood cell count
Haemoglobin
Haematocrit
Mean cell volume
Red blood cell distribution width
Mean cell haemoglobin concentration
Mean cell haemoglobin
Reticulocyte count
Platelet count
White blood cell count
Neutrophil count
Lymphocyte count
Monocyte count
Eosinophil count
Basophil count
Large unclassified cells
Activated partial thromboplastin time
Fibrinogen
Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 4 (males), ~ Day 4 of lactation (females)
- Animals fasted: No
- How many animals: 5 animals per sex per dose group
- Parameters examined:

Urea
Glucose
Aspartate aminotransferase
Alanine aminotransferase
Alkaline phosphatase
Creatine phosphokinase
Lactate dehydrogenase
Sodium
Potassium
Chloride
Total protein
Albumin
Globulin
Albumin/globulin ratio
Cholesterol
Creatinine
Total bilirubin
Calcium
Phosphate

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Weekly
- Dose groups that were examined: All
- Battery of functions tested:
Cageside and standardised arena observations (Weekly)
Grip strength, pain perception, landing foot splay and motor activity (Males (all): Week 4: Females (5 animals per group): Day 3 of lactation)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

Reproductive endpoints included in separate IUCLID summary.

Statistics:

Where required to assist with interpretation, tests were applied to determine the statistical significance of observed differences between Control and groups receiving test item. All statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group.

Body weight and food consumption (prior to mating), haematology, coagulation, clinical chemistry and selected FOB and motor activity data was analysed for homogeneity of variance using the ‘F Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test ie pairwise comparisons were made only if the overall F test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).

Organ weight data was analysed as above, and by analysis of covariance (ANCOVA) using terminal body weight as the covariate.

Clinical signs:

no effects observed

Description (incidence and severity):

9 female animals were found dead/prematurely euthanised either in late gestation/early lactation; the distribution had no assocaiation to dose level and they were not considered test-item related. Excess salivation noted post dose in test animals.

Mortality:

no mortality observed

Description (incidence):

9 female animals were found dead/prematurely euthanised either in late gestation/early lactation; the distribution had no assocaiation to dose level and they were not considered test-item related. Excess salivation noted post dose in test animals.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not specified

Description (incidence and severity):

No inter-group differences were noted during the study, however there was no positive data recorded

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean liver weights were higher than Control in males at all dose levels and slightly higer at 75 and 125 mg/kg/day in females. Mean kidney weights were higher than Control in males at all dose levels (no effects in females).

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: centrilobular hypertrophy (males and females at 125 mg/kg/day: only dose level examined). Kidneys (males at 125 mg/kg/day only): intraepithelial hyaline droplets, single cell necrosis, foci of basophilic (regenerating) tubules, granular casts.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortaility:

There were 9 female animals found dead or prematurely euthanised either in late gestation or early lactation (between Day 22 of gestation and Day 2 of lactation). Although a definitive cause of death could not be established for many of these animals, the distribution of the deaths; 2/10, 4/10, 1/10 and 2/10 (0, 30, 75 and 125 mg/kg/day, respectively) indicated that they were not related to the test item.

Clincal Signs:

Excess salivation was noted post dose in animals receiving the test item. This sign was transient and was considered not to be adverse.

Organ Weights:

Absolute mean liver weights were higher than Control in males at all dose levels (1.11X, 1.06X and 1.13X Control mean, at 30, 75 and 125 mg/kg/day, respectively. In females, absolute mean liver weights were slightly higher than Control at 75 and 125 mg/kg/day (1.06X and 1.08X Control mean, respectively).

Absolute mean kidney weights were higher than Control in males at all dose levels (1.08X, 1.08X and 1.19X Control mean, at 30, 75 and 125 mg/kg/day, respectively). There were no clear differences between the kidney weights of females receiving the test item and Control females.

Histopathology:

In the liver minimal or mild centrilobular hypertrophy of hepatocytes was present in 4/5 males and 5/5 females at 125 mg/kg/day.

In comparison to Control there was a moderate or marked increase in intraepithelial hyaline droplets in the cortical tubules in the kidneys of males at125 mg/kg/day. Associated with the droplets were minimal single cell necrosis of the tubular epithelium, an increase in the incidence and severity of foci of basophilic (regenerating) tubules and, in 2/5 animals, granular casts at the cortico-medullary junction. These findings were considered to be consistent with a diagnosis of ‘hyaline-droplet nephropathy’ caused by an overload of the male rat specific protein alpha-2-microglobulin.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects considered adverse or relevant to humans were observed after 28 or more days of exposure. The highest dose administered was established as the NOAEL

Critical effects observed:

not specified

Conclusions:

Under the conditions of this study, and based on the lack of human relevance of findings in the male kidneys at the high dose, the systemic or parental NOAEL was considered to be 125 mg/kg bw/day.

Executive summary:

A combined repeated dose toxicity study with the reproduction / developmental toxicity screening test was conducted to determine the toxicity to reproduction and/or development of test substance, according to OECD Guideline 422, in compliance with GLP. Sprague-Dawley rats were randomised into 3 test groups and one Control group, each containing 10 males and 10 females. Males were treated by oral gavage at doses from 30, 75 and 125 mg/kg bw/day for 2 weeks prior to mating, then through mating, until the day prior to necropsy (4 weeks of treatment). Females were treated with the same doses for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation (ca. 6 weeks of treatment). The following parameters and end points were evaluated in this study: clinical signs, body weights, food consumption, ophthalmology, detailed functional tests and observations, reproductive parameters (mating and pregnancy performance, fertility, maternal care and pup performance), clinical pathology parameters (haematology, coagulation and clinical chemistry), organ weights, and gross and microscopic pathological examinations. There were no test substance related effects on body weights, body weight changes, food consumption, ophthalmology, detailed functional tests and observations, clinical pathology parameters (haematology, coagulation and clinical chemistry) or gross necropsy findings. Excess salivation noted transiently post dose in animals receiving the test substance may have been a consequence of an unpleasant taste associated the test substance; it was nevertheless considered not to be adverse. The distribution of the 9 female animals found dead or prematurely euthanised (2/10, 4/10, 1/10 and 2/10 at 0, 30, 75 and 125 mg/kg bw/day, respectively) either in late gestation or early lactation (between Day 22 of gestation and Day 2 of lactation) indicated no clear relationship to the test substance. Furthermore, these animals were not observed to be suffering from clinical signs that were considered to be test substance-related; indeed other than the salivation noted above, there were no clinical signs in the animals surviving to the end of the study that were considered to be test substance related. Similarly, test substance related histopathological findings were not observed to a greater degree/severity in the premature decedents relative to the animals surviving to terminal sacrifice. There were no test substance-related effects on mating, pregnancy performance, fertility or maternal care and pup performance (litter survival and pup weights). It should be noted that there were high absolute numbers of litter losses (2/8, 2/6, 1/9 and 3/9 of surviving females at 0, 30, 75 and 125 mg/kg bw/day, respectively) and low viability rates (66%, 74%, 87% and 63% at 0, 30, 75 and 125 mg/kg bw/day, respectively); however the distribution of these findings indicated no clear relationship to the test substance. The minimal or mild centrilobular hypertrophy of hepatocytes in the livers of animals at 125 mg/kg bw/day, correlated with high liver weights in animals receiving the test substance. Whilst this finding was considered to be test substance related, it was not associated with any changes in clinical chemistry parameters and was considered to be an adaptive response and not adverse. The histopathological findings in the kidneys of males at 125 mg/kg bw/day were consistent with a diagnosis of ‘hyaline-droplet nephropathy’, which correlated with high kidney weights in males receiving the test substance. The findings were considered to be caused by an accumulation of complexes of the male rat specific protein alpha-2-microgloblin and the test substance that were resistant to degradation. As little or no alpha-2-microgloblin protein is present in man, Greaves (2007), these findings were considered not to be relevant to man. Therefore, under the conditions of this study, and based on the lack of human relevance of findings, the parental, reproductive and neonatal NOAEL was considered to be 125 mg/kg bw/day (Wallace, 2013).