2-ethylhexylamine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Reference 2

Reference Type:

other company data

Title:

Unnamed

Year:

2005

Report date:

2005

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)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Octylammonium chloride

EC Number:

205-574-8

EC Name:

Octylammonium chloride

Specific details on test material used for the study:

- Purity test date: 2005-05-11
- Lot/batch No.: A0212784

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

Crl:CD®(SD)IGS BR rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 81 days
- Weight at study initiation: 355.2 - 450.2 grams (males) and 196.3 - 309.0 grams (females)
- Fasting period before study: animals from which blood samples were taken were fasted 15 h prior sampling
- Housing: individually during non-mating periods
- Diet: PMI Nutrition International, Certified Rodent LabDiet 5002 ad libitum, except when fasted
- Water: ad libitum
- Acclimation period: 28 days

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- mixing of test substance with NanoPure water
- pH was adjusted to values between 4.0 an 6.0 prior to dosing
- Storage: refrigerated up to 7 days of the low, intermediate,and high dose solution
- Dose volume: 4 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: sperm in vaginal smear; referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): singly

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing samples were diluted to a concentration of 0.100 mg/mL; the pH was adjusted with 8 N NaOH, the samples were then extracted with toluene containing decylamine as internal standard. Duplicate sample analysis was performed using Gas Chromatography.

Duration of treatment / exposure:

Males 47-48 days; females 42-46 days

Frequency of treatment:

daily

Details on study schedule:

- Age at mating: 13 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

Remarks:

This dose were lowered by test day 14 to 100 mg/kg bw/day.

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were based on the results of two previously-conducted range-finding studies.

In the first range-finding study, the test substance was administered as a solution in water
(pH adjusted to 4-6) at daily dose levels of 0, 100, 200, 500, or 1000 mg/kg/day to groups of
5 rats per sex. Doses were administered at a volume of 4 mL/kg. A single dose of

1000 mg/kg/day produced marked excessive toxicity and therefore, this dose level was
terminated after a single dose. The study was continued for a total of four consecutive days and
terminated following persistent dose-related toxicity at the lower levels.

At 1000 mg/kg/day, all five male and five female rats exhibited test substance-related clinical
observations within minutes of dosing. These clinical observations included convulsions,
salivation, splayed hind limbs, ataxia, noisy respiration, loss-of-righting reflex, pallor, facial
staining, and/or hyperreactivity. Three of five males and four of five females were either found
dead or sacrificed in extremis after administration of a single dose. All eight animals that died
after the first dose were observed with gross lesions of the stomach lining including ulceration(s)
and/or discoloration. Based on this marked toxicity, the three surviving animals were not
administered any subsequent doses; this dose level was removed from the study.
The study was continued evaluating doses of 100, 200, and 500 mg/kg/day for a total of four
consecutive days before the study was terminated in its entirety due to excessive toxicity.

At 500 mg/kg/day, doses were administered to males for four consecutive days and to females
for three consecutive days. Test substance-related clinical observations in all animals at this
level included lung noise, salivation, perineal staining, hyperreactivity and/or loss-of-righting
reflex. Two of five males were found dead on test day 4 and four of five females were found
dead on test days 1 through 4 (in the female found dead on day 3, postmortem findings indicative
of a potential intubation error were reported and likely contributed to death in this animal). In
addition to adverse clinical observations, these animals generally lost weight. In males over test days 1-5,
there was a mean weight loss of 35 g compared with a mean gain of 27 g in controls.
In females for the same time period, there was a mean weight loss of 19 g compared with a mean
gain of 9 g in controls. Stomach lesions similar to those reported at 1000 mg/kg/day were
observed in four of the six animals that were found dead at this level.

At 200 mg/kg/day, all five females survived four doses. Three of five males survived four doses;
one male was sacrificed in extremis on test day 4 and the other was found dead on test day 3
(in this male, injuries consistent with an intubation injury that likely contributed to this early
death were evident). Clinical observations related to the test substance observed at this level
included lung noise, gasping, stained fur, salivation, and/or noisy respiration. There were no test
substance-related gross necropsy observations. Test substance-related effects on body weight
parameters were evident in males and females and from test days 1 to 5, male rats gained 74%
less weight than controls and female lost an average of 2 g compared with a gain of 9 g in
controls.

At 100 mg/kg/day, all males and females survived four doses. Clinical observations similar to
those reported for the higher levels occurred with a frequency and onset that was dose-related.
The signs included sporadic occurrences of lung noise, salivation, hyperreactivity. Effects on
body weight gain were evident in males from days 1 to 5 and were 25% lower than for controls
whereas, in females, body weight gains at this level were comparable for the same time period.
There were no test substance-related gross necropsy observations.

Based on these data, doses of 0, 3, 10, 30, and 100 mg/kg/day were selected for a subsequent
range-finding study. In the subsequent study, groups of rats (5 per sex per group) were
administered solutions of octylamine hydrochloride in water (pH adjusted to between 4 and 6;
dose volume of 4 ml/kg) for 14 consecutive days. All animals survived until scheduled
euthanasia and a review of the in-life data (body weights and weight gains, food consumption,
and clinical observations) indicated no evidence of test substance-related toxicity. There were
also no test substance-related gross postmortem observations.
These data suggest that the dose-response curve between the doses of 100 and 200 mg/kg/day is
potentially steep and therefore, the high dose level for the current study was set at
150 mg/kg/day. Doses of 37.5 and 75 mg/kg/day were subsequently selected as the low and
intermediate dose levels to provide two-fold spacing between the dose levels.

Effective test day 14 (November 2, 2005), the high-dose level was lowered from 150 to
100 mg/kg/day and the test formulation concentration was lowered from 37.5 to 25 mg/mL. The
high-dose level was lowered after mortality was produced at 150 mg/kg/day in one male and two
female rats after a minimum of eight consecutive daily doses.

Examinations

Parental animals: Observations and examinations:

CLINICAL SIGNS AND MORTALITY
All animals were observed for mortality twice daily. Clinical observations were done once a day on all animals and detailed observations on day -1, and weekly thereafter on all animals. Observations included (but were not limited to) evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, and unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic, tonic, stereotypical or bizarre behavior.

BODY WEIGHT
The animals were weighed once before first treatment (baseline) and weekly thereafter.

HEMATOLOGY
On test days 13 (males) and 14 (females) blood was collected from orbital sinus of all fasted F0 animals under carbon dioxide anesthesia for hematology; blood for coagulation was collected from the abdominal vena cava of all F0 animals at sacrifice under CO2 anesthesia at study termination (day 47-48 for males and day 42-46 for females).
Following parameter were investigated: red blood cell count, hemoglobin, hematocrit, mean corpuscular (cell) volume, mean corpuscular (cell) hemoglobin, mean corpuscular (cell) hemoglobin concentration, red cell distribution width, absolute reticulocyte count, platelet count, white blood cell count, differential white blood cell count, microscopic blood smear examination, prothrombin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY
On test days 13 (males) and 14 (females) blood was collected from orbital sinus of all fasted F0 animals under carbon dioxide anesthesia for clinical chemistry. Following parameter were investigated: alanine aminotransferase, aspartate aminotrasferase, sorbitol dehydrogenase, alkaline phosphatase, total bilirubin, urea nitrogen, creatinine, triglycerides, cholesterol, glucose, total protein, albumin, globulin, inorganic phosphate, calcium, sodium, potassium, chloride, total bile acids.

URINALYSIS
Urine of fasted animals was collected on day 13 (males) and 14 (females) in metabolism cages. Following parameters were investigated: quality, color, clarity, volume, osmolality, pH, glucose, ketone, urobilinogen, bilirubin, blood, protein, microscopic urine sediment.

FUNCTIONAL OBSERVATIONAL BATTERY AND MOTOR ACTIVITY MEASUREMENT
An abbreviated functional observational battery (forelimb and hindlimb grip strength, open field observations and motor activity) was conducted on all animals prior to initiation of test substance administration on day -8 (males) and day -7 (females) and prior to the end of the premating period on day 12 (males) and day 13 (females). Body weights were collected on the day of neurobehavioral assessments. Each evaluation was performed in 4 replicates.

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, viability at day 4 post partum, clinical observations, weight gain

GROSS EXAMINATION OF DEAD PUPS:
yes, on all surviving pups as well as pups that did not survive to scheduled sacrifice.

Postmortem examinations (parental animals):

NECROPSY
On necropsy animals were macroscopically investigated and following organs of parental animals were weighed: liver, kidneys, adrenal glands, thymus, spleen, brain, heart, testes, epididymides, ovaries.

HISTOPATHOLOGY
Histopathology was performed on following organs of 5 randomly selected animals/sex from the control and high dose group: liver, duodenum, jejunum, ileum, cecum, colon, rectum, kidneys, urinary bladder, spleen, thymus, lymph nodes (mandibular and mesenteric), Peyer's patches, bone marrow (femur), thyroid gland, adrenal glands, brain (including cerebrum, cerebellum, medulla pons), spinal cord (including cervical, mid-thoracic and lumbar), sciatic nerve, femur as well as any organs with gross pathological lesions in all dose groups (trachea).
In addition following reproductive organs from all animals in the control and high dose groups were histopathologically examined: testes, epididymides, prostate, seminal vesicles, coagulation glands, ovaries, oviducts, uterus, cervix and vagina. The presence and number of uterine implantation sites and ovarian corpora lutea were evaluated for all cohabited females.

Postmortem examinations (offspring):

NECROPSY
Macroscopic investigations were also performed on all surviving pups as well as pups that did not survive to scheduled sacrifice. However, organ weight of pups were not assessed.

HISTOPATHOLOGY
No histopathological examinations were performed on pups.

Statistics:

Statistical methods:
- Primarily, Levene's test for homogeneity, one-way analysis of variance, Dunnett's test, Kruskal-Wallis test, Dunn's test and Cochran-Armitage test for trend

Reproductive indices:

Gestation length, reproductive function indices, corpora lutea counts.
The following table lists the indices of reproductive function indices that were calculated for the P1 animals.

Reproductive Function Calculations
Mating Index (%) = (Number copulated a/ Number cohabited) x 100
Fertility Index (%) = (Number pregnant b/ Number copulateda) x 100
Gestation Index (%) = (Number of litters with at least one live pup / Number of litters) x 100
Implantation Efficiency (%)c= (Number of pups born / Number of implantation sites) x 100


a: Evidence of copulation = intravaginal copulatory plug, sperm in vaginal lavage, uterine implantation sites, or delivery of a litter.
b: Including those found dead pregnant during gestation.
c: Determined for each litter. Mean and standard deviation for each dose level were calculated.
d: Excluding litters sacrificed due to death of dam during lactation.

Offspring viability indices:

Pups Born Alive (%)c= (Number of pups born alive / Number of pups born) x 100
0-4 Day Viability (%)c,d= (Number of pups alive day 4/ Number of pups born alive) x 100

c: Determined for each litter. Mean and standard deviation for each dose level were calculated.
d: Excluding litters sacrificed due to death of dam during lactation.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related clinical observations occurred at 75 and 150/100 mg/kg/day. There were no test substance-related clinical observations at 37.5 mg/kg/day in either males or females during any portion of the study.
In males, salivation was observed in 7/12 and 12/12 animals at 75 and 150/100 mg/kg/day, respectively. Additional clinical observations considered to be test substance-related at 150/100 mg/kg/day included irregular respiration, lung noise, and fur-staining (face and nose).
One male rat at 150/100 mg/kg/day exhibited diarrhea, gasping, hunched-over posture, weakness, and abnormal gait/mobility.
In females during the premating period, 1/12 and 12/12 females at 75 and 150/100 mg/kg/day, respectively, were observed with salivation. During gestation, test substance-related clinical observations in females were limited to salivation in 2/12 animals. There were no test substance related clinical observations observed at any dose level during lactation.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two female rats and one male rat from this group were found dead on test days 8 to 12.
One high-dose male (Animal Number 711) was found dead (FD) on day 10. The cause of death
was not determined but was most likely a dosing accident. Although tracheal lesions (minimal to
mild tracheal mucosal inflammation, metaplasia, and hyperplasia) indicated that some dosing
material entered the trachea, histopathology of the lung did not confirm that death was due to a
dosing accident or aspiration.
Two high-dose female rats (Animal Number 808 and Animal Number 810) died on days 8 and
12, respectively. In both cases, there was severe ulceration of the tracheal mucosa with moderate
to severe tracheal inflammation. One of the decedents (Animal Number 810) also had severe
ulcerative bronchitis and aspiration pneumonia. The diagnosed cause of death for both rats was a
dosing accident.
Since all three study deaths were in high-dose rats, it is likely that the test substance played a role
in the mortality however, since the deaths required either accidental intratracheal dosing or
aspiration of esophageal contents, the deaths were not test substance-related in the context of a
gavage study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

P1 Males
Test substance-related and/or statistically significant reductions in mean body weights and body weight gains were observed in males at all dose levels tested. Reductions in these parameters were evident during the first week of dosing and persisted until the end of the study. Mean final body weights were 6, 6, and 12% lower than controls at 37.5, 75, and 150/100 mg/kg/day, respectively, resulting from cumulative body weight gain (test days 1 to 43) reductions of 17, 20, and 43% at the same respective dose levels.

P1 Females
Test substance-related and/or statistically significant effects on body weights and weight gains in females occurred at all levels but these reductions were not as pronounced or dose-dependent as the reductions reported for males. During the premating period, there were slightly (not statistically significant) reduced body weight gains of 48, 27, and 41% at 37.5, 75, and 150/100 mg/kg/day, respectively, that resulted in mean body weights at the end of the premating period that were 2, 3, and 2% lower than controls for the same respective doses. During the gestation period, the effects on body weights and weight gains were more obvious. Mean body
weight gains were significantly lower during the gestation period at all dose levels tested; cumulative gain (gestation days 0 to 21) during gestation was 14, 10, and 11% lower than for controls at 37.5, 75, and 150/100 mg/kg/day, respectively. These reductions during gestation
resulted in mean body weights at the end of gestation (gestation day 21) that were 8, 6, and 7% lower than controls. There were no test substance-related effects on body weights or weight gains during lactation (lactation days 0 to 4) at any dose level tested.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

P1 Male Rats
In male rats, the test substance-related and/or statistically significant effects on food consumption were generally consistent with the effects reported for body weights and weight gains. Overall mean food consumption during the premating period (test days 1 to 15) was 6, 8, and 13% lower than for controls at 37.5, 75, and 150/100 mg/kg/day, respectively.

P1 Female Rats
In female rats, the test substance-related and/or statistically significant effects on food consumption were generally consistent with the effects reported for body weights and weight gains. Mean food consumption values were slightly lower than controls during the premating
period; overall mean food consumption during test days 1 to 15 was 8, 9, and 8% lower that controls at 37.5, 75, and 150/100 mg/kg/day, respectively. The test substance-related reductions were most pronounced during the gestation period; mean food consumed over gestation days 0 to 21 was 12, 12, and 9% lower than controls at 27.5, 75, and 150/100 mg/kg/day. There were no test substance-related effects on food consumption during lactation days 0 to 4.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse changes in hematologic parameters in male or female rats. The following
statistically significant changes in mean hematologic parameters were not adverse or not related
to exposure to compound:
• Hemoglobin concentration was minimally decreased in females dosed with
37.5 mg/kg/day. This change was observed only at the lowest dose level and is
considered unrelated to test substance administration.
• Mean cell hemoglobin concentration was minimally decreased in females dosed with
150/100 mg/kg/day (mean was 99% of the control group mean). There were no
correlative changes in any other red blood cell parameters. This extremely small change
was considered unrelated to treatment.
• Eosinophils were mildly increased in males dosed with 150/100 mg/kg/day (mean was
200% of the control group mean). Eosinophil counts of males in the other groups were
similar and ranged from 0.06 to 0.25x103/uL, but 5 of the 11 counts of
150/100 mg/kg/day males were greater than 0.25x103/uL suggesting a relationship to
treatment. However, there were no other changes in any other white cell parameter, and
no correlative histologic changes in any organ. Therefore, although this change may
have been related to treatment, it is considered non-adverse.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

There were no test substance-related effects or statistically significant differences on forelimb grip strength, hindlimb grip strength, or behavioral observations in the open field in either males or females administered any dosage of the test substance.

Duration of movement and number of movements were decreased in 150/100 mg/kg/day males, and duration of movement was decreased 150/100 mg/kg/day females. Total duration of movement was 20% and 16% lower compared to the control value in 150/100 mg/kg/day males
and females, respectively (not statistically significant). In addition, total number of movements, for 150/100 mg/kg/day males was significantly (22%) lower compared to the control value, and number of movements during the 5th 10-minute interval were significantly lower for 150/100 mg/kg/day males compared to controls. Since there were no test substance-related changes in grip strength, response to tail pinch, approach and touch, and auditory stimulus parameters, and since mortality, effects on body weight, food consumption, and clinical observations occurred at the highest dose level, the lower motor activity may be secondary to systemic toxicity.
There were no test substance-related effects on duration of movement or number of movements in males or females administered dosages of 75 mg/kg/day or below.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Dosing-related microscopic findings were observed in the trachea (mucosal metaplasia,
hyperplasia, and inflammation) of males (≥37.5 mg/kg/day) and females (≥75 mg/kg/day).
However, these tracheal lesions were interpreted to be the result of inadvertent direct exposure of
the trachea to the test substance during dosing and not the effect of gavage exposure. Therefore,
these microscopic findings were not test substance-related in the context of a gavage study.

Squamous metaplasia of the proximal to mid-cervical trachea was observed in more than half of
the male (18/23) and female (11/22) rats given ≥75 mg/kg/day and in one-third (4/12) of the
male rats given 37.5 mg/kg/day of the test substance. All cases were graded as minimal (grade 1
of 4), except for two males (one each at 75 and 150/100 mg/kg/day) that were graded as mild
(grade 2 of 4).
The metaplasia consisted of the partial replacement of the normal ciliated pseudostratified
columnar epithelium (i.e., respiratory epithelium) on the ventral and lateral aspects of the
tracheal mucosa with deciliated round and/or flattened epithelial cells resembling mucous
membrane. In two males (one each at 37.5 and 75 mg/kg/day), the metaplasia was associated with minimal hyperplasia. In two females (one each at 75 and 150/100 mg/kg/day), the
metaplasia was associated with minimal inflammation.
The minimal to mild squamous metaplasia, and associated hyperplasia and inflammation, was
probably the result of transient topical exposure of the tracheal mucosa to the test substance
following gavage administration. These minimal to mild lesions in surviving rats contrast with
the moderate (grade 3 of 4) to severe (grade 4 of 4) tracheal ulceration and inflammation
observed in the two decedent females that died following accidental administration of the test
substance to the trachea.
The minimal to mild metaplasia observed in the surviving rats was interpreted to be a nonadverse
adaptive response, most likely reversible, which was due to transient esophageal reflux or
imperfect dosing. The four incidences of minimal tracheal inflammation or hyperplasia were
also considered to be artifacts of the dosing procedure rather than test-substance related effects.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

One neoplasm occurred in this study. A malignant renal mesenchymal tumor was observed in a
kidney from a high-dose female rat (Animal Number 805). Although this is an uncommon
neoplasm, the individual occurrence was considered to be incidental and not test substance
related.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on mating and fertility (mating and fertility indices,
gestation length, implantation site and corpora lutea counts, and implantation efficiency) at any
level tested; data were comparable across all groups on study. The mean implantation efficiency,
number of pups born, born alive, and % pups born alive in the 150/100 mg/kg/day group were
slightly lower than controls as a result of one small litter that did not survive to day 4. Since
whole litter loss occurs occasionally in rats at low incidences, even in controls, this finding was
considered spurious.

In conclusion, there were no test substance related effects on mating, fertility, and reproductive performance nor were any effects observed
in any of the data collected for F1 offspring (litter sizes, sex ratio, pup weights, and pup clinical observations).

Details on results (P0)

EFFECTS BY DOSE LEVEL

Low dose group: 37.5 mg/kg bw/day:
F0 parental animals
MORTALITY
- no mortality was seen at this dose level
CLINICAL EXAMINATIONS/CLINICAL CHEMISTRY/HEMATOLOGY/URINALYSIS/PATHOLOGY
-statistically significant reductions in body weights (-6%) and body weight gain (-17%) in males from the first week to the end of the study
- slight (not statistically significant) reductions in body weights (-2%/-8%) and body weight gain (-48%/-14%) in females as compared with controls, during premating and gestation, respectively.
-statistically significant reductions in food consumption (-6%) in males during premating
-slight (not statistically significant) reductions in food consumption in females during premating (-8%) and gestation (-12%)
-statistically significant decrease in hemoglobin concentration on day 14 in females, minimal (-4%), not dose-related
-minimal tracheal lesions in males, dosing-related, likely the result of topical exposure to the test substance. Further details on microscopic findings are given at any other informations on results.


Intermediate dose group, 75 mg/kg bw/day:
F0 parental animals
MORTALITY
- no mortality was seen at this dose level
CLINICAL EXAMINATIONS/CLINICAL CHEMISTRY/HEMATOLOGY/URINALYSIS/PATHOLOGY
-salivation in 7/12 males and in 1/12 females during premating
-statistically significant reductions in body weights (-6%) and body weight gain (-20%), as compared with controls, in males from the first week to the end of the study
-slight (not statistically significant) reductions in body weights (-3%/-6%) and body weight gain (-27%/-10%) as compared with controls, in females during premating and gestation, respectively
-statistically significant reductions in food consumption (-8%) in males during premating
-slight (not statistically significant) reductions in food consumption in females during premating (-9%) and gestation (-12%)
-minimal tracheal lesions in males and females, dosing-related, likely the result of topical exposure to the test substance. Further details on microscopic findings are given at any other informations on results.


High dose group, 150/100 mg/kg bw/day:
F0 parental animals:
MORTALITY
Two female rats and one male rat in the 150 mg/kg/day group were found dead on test days 8 to 12. Autopsy suggested a dosing accident aggravated by the irritation from the test substance.
CLINICAL EXAMINATIONS/CLINICAL CHEMISTRY/HEMATOLOGY/URINALYSIS/PATHOLOGY
-salivation in 12/12 male animals, in 12/12 females during premating, and in 2/12 females during gestation
-irregular respiration, lung noise, and fur-staining (face and nose). Diarrhea, gasping, hunched-over posture, weakness, and abnormal gait/mobility in one male
-statistically significant reductions in body weights (-12%) and body weight gain (-43%) as compared with controls, in males from the first week to the end of the study
-slight (not statistically significant) reductions in body weights (-2%/-7%) and body weight gain(-41%/-11%) as compared with controls, in females during premating and gestation, respectively
-statistically significant reductions in food consumption (-13%) in males during premating
-slight (not statistically significant) reductions in food consumption in females during premating (-8%) and gestation (-11%)
-minimal to mild tracheal lesions in males and females, dosing-related, likely the result of topical exposure to the test substance. Further details on microscopic findings are given at any other informations on results.
-statistically significant decrease in mean cell hemoglobin concentration (-1%) on day 14 in females, without associated changes in other white blood cell parameters, not treatment-related
-statistically significant increase in eosinophils (200%) on day 13 in males, without associated changes in other white blood cell parameters or correlative histologic changes, possibly treatment-related, but not considered adverse
-statistically significant decrease in absolute heart weight (-13%) as compared with controls, in males accompanied by slight (not significant) decreases in most other absolute and relative organ weights in males and females, all without microscopic changes and probably related to overall decrease in body weights
- statistically significant increase in relative adrenal gland (36%) and testes (14%) weights in males, without microscopic changes
- a malignant renal mesenchymal tumor was seen in a male. Although this is an uncommon neoplasm, the individual occurrence was considered to be incidental and not test substance related.

REPRODUCTIVE PERFORMANCE
No statistically significant changes in gestation length, reproductive function indices and corpora lutea counts were seen in males and/or females in either dose.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive performance

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects on reproductive performance were seen.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: loss of body weight

Treatment related:

yes

Dose response relationship:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

No statistically significant changes in litter observations like number of live/dead pups at birth, sex ratio, viability at day 4 p.p. and pup weights were seen in the offspring in either dose.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects on live birth index; litter size; pup weight; sex ratio; survival index of pubs were seen. No signs of systemic toxicity were seen.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Microscopic Findings: Dosing-related microscopic findings were observed in the trachea (mucosal metaplasia, hyperplasia, and inflammation) of males and females at all dose levels. However, these tracheal lesions were interpreted to be the result of inadvertent direct exposure of the trachea to the test substance during dosing and not the effect of gavage exposure. Therefore, these microscopic findings were not test substance-related in the context of a gavage study. A summary of effects seen in the trachea of male and female rats on microscopy are given in table 1. All other microscopic observations were consistent with normal background lesions in rats of this age and strain.

 

Table 1: Incidences of Dosing-Related Microscopic Findings in Male and Female Rats

	Male				Female
Dose (mg/kg bw/day)	0	37.5	75	150/100	0	37.5	75	150/100
Number of rats at sacrifice	12	12	12	11	12	12	12	10
Trachea
Metaplasia, mucosal	1	4	11	7	1	1	5	6
Hyperplasia, mucosal	0	1	1	0	0	0	1	0
Inflammation, mucosal	0	0	0	0	0	0	1	1

 The authors interpreted the underlined to be dosing-related increases in microscopic findings.

  

Trachea

Squamous metaplasia of the proximal to mid-cervical trachea was observed in more than half of the male (18/23) and female (11/22) rats given >/= 75 mg/kg/day and in one-third (4/12) of the male rats given 37.5 mg/kg bw/day of the test substance. All cases were graded as minimal (grade 1 of 4), except for two males (one each at 75 and 150/100 mg/kg bw/day) that were graded as mild.

The metaplasia consisted of the partial replacement of the normal ciliated pseudostratified columnar epithelium (i.e., respiratory epithelium) on the ventral and lateral aspects of the tracheal mucosa with deciliated round and/or flattened epithelial cells resembling mucous membrane. In two males (one each at 37.5 and 75 mg/kg bw/day), the metaplasia was associated with minimal hyperplasia. In two females (one each at 75 and 150/100 mg/kg bw/day), the metaplasia was associated with minimal inflammation.

 The minimal to mild squamous metaplasia, and associated hyperplasia and inflammation, was probably the result of transient topical exposure of the tracheal mucosa to the test substance following gavage administration. These minimal to mild lesions in surviving rats contrast with the moderate (grade 3 of 4) to severe (grade 4 of 4) tracheal ulceration and inflammation observed in the two decedent females that died following accidental administration of the test substance to the trachea.

The minimal to mild metaplasia observed in the surviving rats was interpreted to be a nonadverse adaptive response, most likely reversible, which was due to transient esophageal reflux or imperfect dosing. The four incidences of minimal tracheal inflammation or hyperplasia were also considered to be artifacts of the dosing procedure rather than test-substance related effects.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, a definitive no-observed-effect level (NOEL)a for
reproductive and neurobehavioral toxicity in P1 adults and effects on F1 offspring growth and
survival was 100 mg/kg/day, the highest dose level tested. A definitive systemic NOEL was not
established for systemic toxicity in P1 males and females since reductions in body weights,
weight gains, and food consumption, as compared with vehicle controls, were observed at all
dose levels tested (≥ 37.5 mg/kg/day).

Executive summary:

A combined repeated dose toxicity study with a reproduction/developmental toxicity screening

test was conducted with octylamine hydrochloride. Crl:CD®(SD)IGS BR rats (12/sex/dose level)

were dosed with the test substance in NanoPure® water (4 mL/kg ) once daily by gavage at dose

levels of 0, 37.5, 75, or 150/100 mg/kg/day. Following a two-week premating period during

which dosing continued on a daily basis, P1 males and females were cohoused for up to 2 weeks

within their respective treatment groups to produce F1 litters. Dams were allowed to deliver and

rear their offspring until postpartum day 4. Careful clinical observations were recorded once

daily during dosing; detailed clinical observations were recorded once during the pretest period

and weekly thereafter. Body weights and food consumption were recorded weekly for P1 males

and females (premating), on days 0, 7, 14, and 21 of gestation and on days 0 and 4 of lactation;

food consumption was not measured during cohabitation or thereafter for males, or for females

with no evidence of copulation. An abbreviated neurobehavioral evaluation consisting of a

functional observational battery and motor activity was conducted in P1 rats once before the

initiation of dosing and again near the end of the premating period. Clinical pathology

parameters were measured in P1 rats near the end of the premating period (hematology, clinical

chemistry, urinalysis) and at terminal sacrifice (coagulation). F1 litter examinations (pup

viability, individual pup weights, clinical observations) were performed at birth and on lactation day 4.

All P1 rats were given a gross pathological examination at terminal sacrifice. Selected tissues

and gross lesions were weighed and/or retained for microscopic examination. Uterine

implantation sites and ovarian corpora lutea were counted in P1 females. A histological

examination of all tissues saved was conducted for all animals in the control and high dose level

groups. Examination of tissues from the remaining groups was limited to relevant gross lesions

and those tissues that demonstrated gavage-related histological changes in the high dose level group.

Test substance-related, adverse effects at 37.5 mg/kg/day and higher were limited to significant

reductions in mean body weight, weight gain, and food consumption. At 75 and

150/100 mg/kg/day, these reductions were accompanied by clinical signs of toxicity.

There was no adverse test substance-related effect on any of the following parameters at any

level tested: reproductive performance (fertility, pregnancy, and reproductive outcome),

neurobehavioral evaluations (functional observational battery and motor activity), clinical

pathology parameters (hematology, clinical chemistry, urinalysis, coagulation), gross and

microscopic anatomic pathology.

Under the conditions of this study, a definitive no-observed-effect level (NOEL) for

reproductive and neurobehavioral toxicity in P1 adults and effects on F1 offspring growth and

survival was 100 mg/kg/day, the highest dose level tested. A definitive NOEL was not

established for systemic toxicity in P1 males and females since reductions in body weights,

weight gains, and food consumption, as compared with vehicle controls, were observed at all

dose levels tested (≥ 37.5 mg/kg/day).