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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
OEDC 422
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-02-21 to 2009-11-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3650
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: Liquid at room temperature
Details on test material:
- Name of test material (as cited in study report): Quarternary ammonium compounds, coco alkylmethylbis(hydroxyethyl)methyl, chlorides
- Physical state: Liquid
- Analytical purity: 75 %
- Lot/batch No.: 7855 19
- Expiration date of the lot/batch: August 2008
- Storage condition of test material: Room temperature (20 ± 5 "C)

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat, HanRcc: WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd
- Age at study initiation: 11 weeks
- Weight at study initiation: 292 to 326g (males) 178 to 212g (females)
- Fasting period before study:
- Housing: Makrolon type-3 cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 (°C)
- Humidity: 30-70 %
- Air changes: 10-15 per hr
- Photoperiod: (12 hrs dark / 12 hrs light)

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
28-days minimum for males
Approximately 7 weeks for females
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation:until copulation was observed
- Proof of pregnancy: vaginal plug / sperm in vaginal smear
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Indivdual samples which were analysed ranged from 90.3 to 117.2% of nominal values (within the 20% acceptance limit).
Duration of treatment / exposure:
28-days minimum for males
Approximately 7 weeks for females
Frequency of treatment:
Daily
Details on study schedule:
see table 1. - study schedule.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control (23% isopropanol in water) dose voolume 10mL/kg
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Remarks:
Tests substance contains 23% ispopropanol dose adjusted for 75% purity dose volume 10mL/kg
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Tests substance contains 23% ispopropanol dose adjusted for 75% purity dose volume 10mL/kg
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Tests substance contains 23% ispopropanol dose adjusted for 75% purity dose volume 10mL/kg
No. of animals per sex per dose:
10 males and 10 females per dose group
Control animals:
yes
Details on study design:
- Dose selection rationale: Dose range finding study using dose levels of 20, 60 and 180 mg/kg/day resulting to be lethal at 180 mg/kg/day and in NOEL of 20 mg/kg/day
- Rationale for animal assignment: random

Control animals:
The control group was treated with a solution of Isopropanol anhydrous (CAS No. 00067-63-0) in water at the same dilution as for the highest dose level formulation.

Examinations

Parental animals: Observations and examinations:
CAGESIDE OBSERVATIONS:
Performed daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: once prior to the first administration of the test substance and weekly thereafter.

BODY WEIGHT:
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION:
- Food consumption for males: recorded weekly during and after pairing period
- Food consumption for females: pre-pairing period days 1-8 and 8-14; gestation days 0-7, 7-14 and 14 to 21 post coitum, and days 1-4 post partum

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood:
males - samples were collected on the day before or on the daz of sheduled necropsy.
females - samples were collected from lactating females 5 days post partum.
- Anaesthetic used for blood collection: light isoflurane anesthesia
- Animals fasted: 18 hours before collection
- How many animals: 5 male and 5 females
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood:
males - samples were collected on the day before or on the daz of sheduled necropsy.
females - samples were collected from lactating females 5 days post partum.
- Anaesthetic used for blood collection: light isoflurane anesthesia
- Animals fasted: 18 hours before collection
- How many animals: 5 male and 5 females
- Parameters checked in table [No.3] were examined.


NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: P-generation males and females were studied before the scheduled sacrifice and on days 3 or 4 post partum, respectively
- Dose groups that were examined: 5 animals per sex per group
- Battery of functions tested: cage side observations/ hand-held observations/ grip strength / rearing behaviou/reflexes /landing foot splay
Oestrous cyclicity (parental animals):
not recorded
Sperm parameters (parental animals):
Parameters examined in P0 male parental generations:
Testis weight, epididymis weight, sperm staging
Litter observations:
Litters were examined for litter size, live births, still births and gross anomalies. The sex ratios of the pups were recorded. Pups were weighed individually (without identification) on days 0, 1 and 4 post partum.
Postmortem examinations (parental animals):
NECROPSY: The following tissue samples were collected; ovaries, prostate, seminal vesicles with coagulating gland, testes, epididymides, brain, spinal chord, small and large intestines, stomach, liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea and lungs, uterus, urinary bladder, lymph nodes, sciatic nerve, bone marrow.
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: all tissues and organs collected at necropsy were examined by histopathlogical techniques.
Postmortem examinations (offspring):
Examined for gross anomilies
Statistics:
The Dunnett-test (many to one t-test) based on pooled variance estimate was applied if the variable could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
Reproductive indices:
Successful breeding pairs, fertility index and conception rate.
Offspring viability indices:
The offspring the viability index was 96.4, 99.3 and 100.0%, in order of ascending dosages. (0, 25 and 50 mg/kg respectively).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 100mg/kg bw/day one male and two females were found dead on day 5 of the pre-paring period, the remaining animals were terminated for ethical reasons. At 50 mg/kg bw/day one female was found dead on day 6 and 4 others were sacrificed due to ethical reasons on day 8.
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
At 100mg/kg bw/day one male and two females were found dead on day 5 of the pre-paring period, the remaining animals were terminated for ethical reasons. At 50 mg/kg bw/day one female was found dead on day 6 and 4 others were sacrificed due to ethical reasons on day 8.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significant reduction in body weight gain in pre-pairing period in the 50mg/kg bw/day dose group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males: at 25 and 50 mg/kg/day mean food consumption was dose-dependently reduced during the first part of the pre-pairing period. Afterwards, no test item-related effects were noted. Females: at 50 mg/kg/day mean food consumption was reduced during the first part of the pre-pairing period. Afterwards, no test item-related effects were noted.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males
No test item—related effects were noted. In group 3, the statistically significantly high level of absolute neutrophilis count (+42.5% compared to the control group) was within the range of the historical reference values.

In groups 2 no altered parameters were observed.

Females
In group 2, the relative count of monocytes was statistically significantly higher (+2l.7% compared to the control group value). This was not considered to be a test item—related effect since the absolute count was not significantly increased and the higher levels of relative and absolute counts of monocytes also observed in group 3 were not statistically significant.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males
In groups 2 and 3, the activity of alanine aminotransferase was dose dependently and statistically significantly increased (+68.7% and +96.8% compared to the control group, respectively). These increases were not accompanied by histopathological findings in the liver.
In group 3, the statistically significantly lower level of total bilirubin and phosphorus (-39.0% and -12.5% versus the control, respectively) were within the range of the reference values. In group 2 the statistically significantly higher levels of cholesterol and sodium (+20.0% and +0.8% compared to the control group values, respectively) were within the range of the historical reference values.

Females
In group 3, the activity of creatine kinase was statistically significantly increased (+38.6% versus the control group); the levels of calcium and phosphorus were statistically significantly increased (+5.6% and +22.l% versus the control group, respectively) and the level of chloride was statistically significantly decreased (-3.0% versus the control group). All these values were within the range of historical control reference values. The activity of alanine aminotransferase was statistically significantly increased (+75.0% versus the control group) and was not accompanied by histopathological findings in the liver.

In groups 2 and 3, the levels of creatine were statistically significantly decreased (-12.6% and - 16.5% compared to the control group, respectively) but within the range of historical control reference values and therefore not considered to be of toxicological relevance.

In group 2, the statistically significantly higher level of calcium (+6.0% compared to control group) was within the range of historical control reference values.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
All animals in group 3 were noted to have ruffled fur during the open field observations. This sign was not considered indicative of any neurological effect, but related to the general poor health status of these animals.

The other parameters such as rearing number and puddles of urine, activity and salivation did not give any indication of a test item-related effect. Common findings such as fearfulness and spontaneous vocalization when the rat was removed from the cage were observed in a single male in the control group.

Mean values of grip strength (fore and hind paws) and landing foot splay gave no indication of test item-related effects. Body temperature in males was statistically significantly lower in group 2 compared to the control group (37.6 °C compared to 38.3 °C in the control group). Since the body temperature in group 3 was not affected this finding was considered to be incidental.

Locomotor activity was assessed quantitatively in terms of low beam counts in an activity monitor. For females in group 3, the locomotor activity was statistically significantly lower during the last 6 minutes of the 30 minutes measurement period, which was considered to be of incidental nature since it was within the range of historical control data. No other statistical significances occurred.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Marked inflammatory lesions in the forestomach observed in animals in the 50 and 100 mg/kg bw/day which resulted in death.

Acute/subacute necrotizing inflammation of the forestomach mucosa was diagnosed in one female in group 2 (moderate), one female in group 3 (slight), and ten males and nine females in group 4 (slight to marked in degree).

Forestomach erosion was noted in one male in group 4.

Hyperkeratosis/acanthosis of forestomach mucosa was noted in five males and five females in group 2 (minimal to marked in degree), five males and six females in group 3 (moderate to marked in degree), and two males and six females in group 4 (slight to moderate in degree). The changes in the stomach were considered to be a local effect consequent to the highly irritating properties of the test item administered as a bolus of gavage.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Under the conditions of this study, the test item did not reveal effects on the completeness of stages or cell populations in any of the treated groups, compared to the control. There was no indication of maturation arrest, re-absorption of sperm or any other degeneration type.
Reproductive performance:
no effects observed
Description (incidence and severity):
Fertility index and conception rate were 100% at 0, 25 and 50 mg/kg/day

Details on results (P0)

Fertility index and conception rate were 100% at 0, 25 and 50 mg/kg/day.
Mean number of corpora lutea per dam was similar in all groups.
The mean duration of gestation was unaffected by exposure to the test substance.
The mean number of implantations per dam was similar in all groups. The total and mean post implnatation loss was not affected by the treatment with the test item.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Moderate necrotizing inflammation of the forestomach mucosa in one out of the five females examined at 25 mg/kg/day, however this is a local effect so it does not prevent the setting of a systemic NOAEL.
Dose descriptor:
NOEL
Remarks:
reproduction
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Considering that there were no reproductive effects in the surviving dams treated with 50 mg/kg/day, and no changes in the male reproductive system were noted, this NOEL may be even higher.

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
25 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
duodenum
ileum
jejunum
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effects on day 1 and day 4 post partum
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No abnormal findings were observed during the macroscopic examination of F1 pups.
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Litter size at first litter check was not affected by the treatment with the test item. Post natal loss was not affected by the treatment of the test substance.

Effect levels (F1)

Dose descriptor:
NOEL
Remarks:
developmental
Generation:
F1
Effect level:
> 25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Could be higher as no adverse effects were seen at 50mg/kg, but only 5 parental females survived against 8 required.

Overall reproductive toxicity

Reproductive effects observed:
no
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
no
Relevant for humans:
no

Any other information on results incl. tables

Reproduction data. summary of performance

 Group (mg/kg/day)  1 (0)  2 (25)  3 (50)  4 (100)
 Female numbers  41 -50  51 -60  61 -70  71 -80
 No. of females paired 10   10  5
 No. females died before scheduled necropsy  -  -  10
 No. females with early delivery  1  0  0  -
 No. females that reared pups until day 4 post partum  10  10  5  
         

Applicant's summary and conclusion

Conclusions:
The NOAEL for reproductive/developmental toxicity was set at the 25mg/kg bw/day dose level. Considering that there were no reproductive effects in the surviving dams treated witih 50 mg/kg bw/day, and no changes in the male reproductive system were noted, the NOAEL may be even higher.
Executive summary:

At 50 mg/kg bw/day, the evaluation of the reproduction/development parameters were carried out only on 5 females and it did not give an indication of any test substance related effect. Due to the number of females available for assessing possible reproductive/development effects, lower than the number required by the relevant guidelines (5 versus 8), it was not possible to definitively set the NOEL for reproductive and developmental toxicity at this dose level.

The overall indication from the study is that the test item will not affect reproduction or fertility in rats and therefore no classification is required.