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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-hydroxyethyl)-3,5,5-trimethylhexanamide

EC Number:

800-884-5

Cas Number:

1154308-86-7

Molecular formula:

C11 H23 N1 O2

IUPAC Name:

N-(2-hydroxyethyl)-3,5,5-trimethylhexanamide

Details on test material:

- Name of test material (as cited in study report): Additol® SXW 6246/90 Coating Additives
- Physical state: Amber Liquid
- Analytical purity: 94.67% (+5.33% water)
- Batch no.: 210129777
- Expiration date of Batch no. 210129777: 28 February 2013
- Storage condition of test material: Ambient, Dark
- Composition of test material, percentage of components: Amides, C8-12, N-(hydroxyethyl) 90622-90-5 (94.67%) and Water 7732-18-5 (5.33%)

Test animals

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on 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

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Up to 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged: Mated females were transferred to individual solid bottomed cages
- Any other deviations from standard protocol: None

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

Details on study schedule:

- Age at mating of the mated animals in the study: 10-11 weeks

Doses / concentrations

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

Examinations

Parental animals: Observations and examinations:

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)

Oestrous cyclicity (parental animals):

During the mating period

Sperm parameters (parental animals):

Not assessed

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals: Following 4 weeks of dosing and after the 2 week mating period.
- Maternal animals: All surviving animals: Between Day 5 and 7 of lactation

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Corpora lutea and implantation counts were completed.

ORGAN WEIGHTS
The tissues indicated below were weighed:

Brain
Epididymis
Gland, adrenal
Gland, pituitary
Gland, prostate
Gland, thyroid
Heart
Kidney
Live
Lung
Ovary
Spleen
Testis
Thymus
Uterus

HISTOPATHOLOGY

The tissues indicated below were prepared for microscopic examination:

Bone marrow, femur
Bone marrow, sternum
Bone, femur
Bone, rib
Bone, sternum
Brain
Cervix
Epididymis
Eye
Gland, adrenal
Gland, harderian
Gland, lacrimal
Gland, mammary
Gland, parathyroid
Gland, pituitary
Gland, prostate
Gland, salivary
Gland, seminal vesicle
Gland, thyroid
Gross lesions
Gut-associated lymphoid tissue
Heart
Kidney
Large intestine, caecum
Large intestine, colon
Large intestine, rectum
Larynx
Liver
Lung
Lymph node, mandibular
Lymph node, mesenteric
Muscle, skeletal
Nasal cavity
Nerve, optic
Nerve, sciatic
Oesophagus
Ovary
Oviduct
Pancreas
Pharynx
Skin
Small intestine, duodenum
Small intestine, ileum
Small intestine, jejunum
Spinal cord
Spleen
Stomach
Testis
Thymus
Tongue
Trachea
Ureter
Urinary bladder
Uterus
Vagina

Postmortem examinations (offspring):

Where practicable, animals F1 pups found dead or killed were sexed, and then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities discarded. Any abnormal pups were fixed in 10% neutral buffered formalin. Externally normal pups were discarded.

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.

Reproductive indices:

The following reproductive indices were evaluated:

For each group:

Fertility Index (male) = Number siring a litter
Number paired

Fertility Index (female) = Number pregnant
Number paired

Gestation Index = Number bearing live pups
Number pregnant

Offspring viability indices:

For each litter and group:

Birth Index = Total number of pups born (live and dead)
Number of implantation scars

Live Birth Index = Number of pups live on Day 0 of lactation
Total number born (live and dead)


Viability Index = Number of pups live on Day 4 of lactation
Number live on Day 0

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

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.

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Details on results (P0)

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.

Mating Performance, Fertility and Duration of Gestation and Litter Size:

Mating performance, fertility indices, corpora lutea counts, implantation counts, and the duration of gestation, were similar between Control females and those receiving the test item.

The birth and the live birth indices were notably lower than Control in the 30 mg/kg/day dose group (both 83% versus 98 and 99% respectively in Control); whilst the live birth index was also notably lower in the 125 mg/kg/day dose group (86% versus 99% in Control). The mean number of pups born per litter and the mean number of live pups born per litter were notably lower than Control in the 30 mg/kg/day dose group (both 0.76X Control mean); however these indices were only slightly lower than Control in the 75 and 125 mg/kg/day dose groups (lowest value 0.90X Control mean).

Given the absence of a clear dose response relationship in these indices, the changes were considered to be incidental.

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.

Effect levels (P0)

Key result

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 adverse effects were observed The NOAEL is the highest dose administered.

Remarks on result:

not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

Externally visible abnormalities only

Histopathological findings:

not specified

Details on results (F1)

Litter Survival, Litter and Pup Weights:

There was a notable number of litter losses in this study; 2/8, 2/6, 1/9 and 3/9 of surviving females receiving 0, 30, 75 and 125 mg/kg/day, respectively. Animal 79 (125 mg/kg/day) suffered a litter loss on Day 0 of lactation and was subsequently prematurely euthanised on Day 2 of lactation (see Section 12.2). Similarly, viability was notably low 66%, 74%, 87% and 63% at 0, 30, 75 and 125 mg/kg/day, respectively. These values were below the lower range of the historical control data (79%) in all but the 75 mg/kg/day dose group. Given the absence of a clear dose response relationship in these indices the changes were considered to be incidental.

Mean litter weights were slightly lower than Control on Day 1 of lactation at 30 mg/kg/day (81% of Control), but mean pup weights were similar to Control and it was considered that the difference in mean litter weights were a consequence of the smaller litter sizes in the 30 mg/kg/day dose group. Mean litter and pup weights were similar between Control and the 75 and 125 mg/kg/day dose groups.

Observations among Dams/Pups:

The type and distribution of observations amongst dams and their litters during lactation did not indicate any association with the test item.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed The NOAEL is the highest dose administered.

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the parental, reproductive and neonatal NOAELwas 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. Refer to the repeated dose section for details on general systemic parameters.

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. Centrilobular hypertrophy of hepatocytes in the livers of high dose animals was considered to be test substance-related but not adverse. The histopathological findings in the kidneys of high dose males were consistent with a diagnosis of hyaline-droplet nephropathy, and this effect has been determined to be not relevant to humans. There occurred some mortality among pregnant females, but as this was also seen in the control group and was not associated with a dose-response relationship, it was not considered test-substance related. 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).