Docosyltrimethylammonium methyl sulphate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

from 2009-07-08 to 2010-01-14

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report which meets basic scientific principles. Justification for read-across see chapter 1 of the chemical safety report.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

no guideline required

Principles of method if other than guideline:

The purpose of this study was to select suitable dosages and appropriate administration method to be used in the subsequent reproduction/developmental toxicity screening test in the Han Wistar rat.

APPLICATION BY GAVAGE
Three groups of 3 males and 3 females were treated by gavage with C20/22-ATQ trocken once daily. Males were treated over a 14 day pre-pairing period and during the pairing period and up to one day before necropsy (for at total of 29 days of treatment). Females were treated throughout the pre-pairing, pairing and gestation period up to day 13 post coitum (for a total of at least 29 days of treatment).

ADMINISTRATION IN THE DIET
C20/22-ATQ trocken was administered in dietary levels of 0, 600, 1800 and 6000 ppm to four groups of 3 males and 3 females:


During the treatment the following observations were recorded: viability/mortality, clinical signs, food consumption, body weights and body temperature. Hematology and clinical biochemistry parameters were determined. Upon termination of the study, during the necropsy males and females were subjected to macroscopical examination. In addition, the number of corpora lutea in each ovary, the number and distribution of implantation sites, live or dead embryos, and early and late embryonic deaths in each uterine horn were recorded during the necropsy of females for evaluation of fertility.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

EC Number:

271-756-9

EC Name:

Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

Cas Number:

68607-24-9

IUPAC Name:

68607-24-9

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Animals: Rat, HanRcc: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
Number of Animals: 21 males (3 per group) and 21 females (3 per group)
Age (at Start of Treatment): 11 weeks
Body Weight Range (at Start of Treatment): Males (293 to 319 g) and females (180 to 204 g)
Identification: Cage card and individual animal number (ear tattoo).
Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). There was 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (¿Lignocel¿J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
Diet: Pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum (batch no. 15/09).
Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.

Administration / exposure

Route of administration:

other: oral by application by gavage (groups 1 - 3) and by feeding (groups 4 - 7)

Vehicle:

water

Remarks:

(by application by gavage in groups 1 - 3)

Details on exposure:

APPLICATION BY GAVAGE (GROUPS 1, 2 AND 3)

DOSE FORMULATIONS
The dose formulations were prepared daily using the test item as supplied by the Sponsor.

C20/22 ATQ trocken was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS
Dose formulations were stored at room temperature (20 ± 5 °C) in brown glass beakers.

TREATMENT
Method: Oral, by gavage (groups 1 - 3)
Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
Frequency of Administration: Daily
Dose Levels by Gavage:
Group 1: 0 mg/kg/day (control group)
Group 2: 50 mg/kg/day
Group 3: 150 mg/kg/day
Rationale for Dose Level Selection: The dose levels were selected by the Sponsor based on a previous 28-day repeated dose toxicity study in rats.
Dose Volume: 10 mL/kg body weight
Duration of Acclimatization Period: 7 days
Duration of Treatment Period: Approximately 6 weeks


ADMINISTRATION IN THE DIET (GROUPS 4, 5, 6 AND 7)

FEED PREPARATIONS
Dietary admixtures were prepared every three weeks using the test item as supplied by the Sponsor.

Fresh batches of the feed pellets for this study were prepared based upon the food consumption and body weights.

C20/22-ATQ trocken was weighed into a tared glass baker on a suitable precision balance, and mixed with microgranulated feed separately for each dose group. An appropriate amount of water was added to aid pelleting. The pellets were dried with air for approximately 48 hours before storage.

Control feed for the animals in group 1 was prepared similarly, but without test item.

STORAGE OF FEED PREPARATIONS
Feed preparations were stored at room temperature (17 - 23 °C) in disposable paper bags until use.

TREATMENT
Method: By feeding (groups 4 - 7)
Frequency of Administration: Daily
Dietary Levels:
Group 4: 0 ppm (control group)
Group 5: 600 ppm
Group 6: 1800 ppm
Group 7: 6000 ppm
Rationale for Dose Level Selection: The dose levels were selected by the Sponsor based on a previous 28-day repeated dose toxicity study in rats.
Duration of Acclimatization Period: 7 days
Duration of Treatment Period: Approximately 6 weeks

Details on mating procedure:

During the pairing period, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if:

- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.

The day of mating was designated day 0 post coitum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSES OF DOSE AND FEED FORMULATIONS

On the first treatment day sample from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (4 hrs and 7 days). The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Analytic Department (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by a light scattering (ELSD) detector following an analytical procedure developed at Harlan Laboratories. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.

Samples for analyses of test item, its content and homogeneity in the feed were drawn at the start of the pre-pairing period and beginning of gestation or end of pairing period (i.e. two occasions). The analyses were performed by Analytic Department (Harlan Laboratories Ltd., Itingen / Switzerland) using a method developed at Harlan Laboratories. Stability (3 weeks) of the test item in the feed were determined at the start of the pre-pairing period (i.e. one occasion).

For assessment of content and homogeneity, a 50 g sample was collected from each the top, middle and bottom of every dietary admixture of the respective diet preparation (from group 1 admixtures only one sample was drawn from the middle of the preparation). Samples for assessment of content and homogeneity were collected on the day of preparation and retained frozen between ca. -24 and -16 °C prior to analysis. For assessment of stability, a 50 g sample was drawn from the middle of the dietary admixture on the day of preparation, stored at room temperature for three weeks and consequently frozen as described above.

The identity of C20/22-ATQ trocken was confirmed in application formulations by gavage and by feeding investigated during the study. The test item was found to be within the accepted range of ±20% with reference to the nominal concentration in all applied formulations.
The homogeneous distribution of C20/22-ATQ trocken in the preparations for gavage and for dietary administration was approved because single results did not deviate more than 11.8% (<15%) from the corresponding mean.

The test item when prepared for gavage was stable when stored for 7 days at room temperature. Maximum variation from time zero (homogeneity) mean was 2.3% and therefore within the accepted limit of 10%.

The test item was considered to be stable in the diet for at least 21 days when kept at room temperature although the result of group 7 stability sample exceeded the accepted limit by 2.1%.

Duration of treatment / exposure:

Approximately 6 weeks

Frequency of treatment:

Daily

Details on study schedule:

Acclimatization 7 days (males and females)
First test item administration Day 1 of pre-pairing (males and females)
Pre-Pairing 14 days (males and females)
Pairing 5 days (males and females)
Treatment Ends:
Gavage On day 13 post coitum (females); on day before sacrifice (males)
Dietary Until sacrifice (males and females)
Necropsy On day 14 post coitum (females); after treatment of at least 28 days, when no longer needed for assessment of reproductive effects (males)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
50 mg/kg bw/day
Basis:
actual ingested
(gavage)

Doses / concentrations 2

Remarks:

Doses / Concentrations:
150 mg/kg bw/day
Basis:
actual ingested
(gavage)

Doses / concentrations 3

Remarks:

Doses / Concentrations:
600 ppm
Basis:
nominal in diet
(administration in the diet)

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1800 ppm
Basis:
nominal in diet
(administration in the diet)

Doses / concentrations 5

Remarks:

Doses / Concentrations:
6000 ppm
Basis:
nominal in diet
(administration in the diet)

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

yes, plain diet

Details on study design:

- Dose selection rationale:
Computer-generated random algorithm.
In addition body weights (recorded on the day of allocation were taken into consideration in order to ensure similar mean body weights in all groups.

Examinations

Parental animals: Observations and examinations:

Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy).
Food Consumption: Males (pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; after pairing period weekly); females (pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; gestation days 0 - 7 and 7 14). No food consumption was recorded during the pairing period.
Body Weights: Recorded daily during the treatment period until termination of the study.
Body Temperature: Recorded on day 5 of the pre-pairing period


CLINICAL LABORATORY INVESTIGATIONS
Blood samples were obtained from all males on day 29 of treatment and from all females on day 14 post coitum from each group. Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The male animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

Additionally serum samples were also collected and stored for further analysis if required by the Sponsor.


HEMATOLOGY
The following hematology parameters were determined in all treatment groups:

Complete Blood Cell Count:
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Leukocyte count, total
Differential leukocyte count
Platelet count

Coagulation:
Prothrombin time (= Thromboplastin time)
Activated partial Thromboplastin time


CLINICAL BIOCHEMISTRY
The following clinical biochemistry parameters were determined:
Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Aspartate aminotransferase
Alanine aminotransferase
Alkaline phosphatase
Gamma-glutamyl-transferase
Bile acids
Sodium
Potassium
Chloride
Calcium
Phosphorus
Protein, total
Albumin
Globulin
Albumin/Globulin ratio


BIOANALYTICAL ASSAYS
Blood samples (approximately 1 mL) were obtained before dosing from all animals on day 1 of the pre-pairing period and on day 5 of the pre-pairing period. Blood samples (approximately 1 mL) were drawn sublingually from all animals under light isoflurane anesthesia and collected in MicrotainerTM tubes and kept at room temperature for ca. 20 minutes, so that the blood clotted. Centrifugation was performed at room temperature (approx. 1900 g, 10 minutes at 20 ± 5 °C). Serum samples were obtained and stored in EppendorfTM tubes at -80 ± 10 °C and shipped on dry ice to to the study scientist of bioanalytical assay part (Bioanalytics Department, Harlan Laboratories Ltd., Füllinsdorf / Switzerland). Samples were stored approximately at -80 °C until work up.

Serum levels of three biomarkers (alpha-acid glycoprotein, TNF-alpha and cortisol) were determined in control and high dose groups by respective photometric Enzyme-Linked Immuno Sorbent Assays.

Postmortem examinations (parental animals):

TERMINATION OF THE STUDY
NECROPSY OF FEMALES
On day 14 post coitum, females were sacrificed by CO2 asphyxiation and necropsied.

For evaluation of fertility, the number and distribution of implantation sites, live or dead embryos, and early and late embryonic deaths in each uterine horn were recorded.

The number of corpora lutea in each ovary was recorded.

Organs showing macroscopic changes were fixed and preserved in neutral phosphate buffered 4% formaldehyde solution against the contingency of histological examination.


NECROPSY OF MALES
Males were sacrificed and necropsied after the pairing period had been completed, following treatment for at least 28 days. Any macroscopic abnormalities were recorded. Organs showing macroscopic changes were fixed and preserved in neutral phosphate buffered 4% formaldehyde solution against the contingency of histological examination.


ORGAN WEIGHTS
The following organs from all males and females which survived until the scheduled necropsy were trimmed from any adherent tissue, as appropriate, and their wet weight taken.

Adrenal glands (weighed as pairs)
Brain
Heart
Kidneys (weighed as pairs)
Liver
Thymus
Spleen


TISSUE PRESERVATION
The following tissues from all parental animals were preserved in neutral phosphate buffered 4% formaldehyde solution or in Bouin¿s fixative for histopathological examination:

Liver
Kidneys
Adrenals
Heart
Brain
Thymus
Spleen
Lung
Mesentheric lymph node
Testes (in Bouin¿s fixative)
Epididymides (in Bouin¿s fixative)
Uterus

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:

- Means and standard deviations of various data were calculated.

- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.

- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.

- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

The calculations for evaluation of the reproduction data were performed by a computer program based on on-line recorded data. The following data were calculated: mean pre-coital time, percentage mating, fertility index, conception rate, pre- and post-implantation losses.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

ruffled fur and/or diarrhea in groups 2, 3, 6 and 7

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced respectively lower in groups 2 and 5

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

reduced respectively lower in groups 2 and 5

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: see "Any other information on results incl. tables"

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

1 APPLICATION BY GAVAGE
1.1 MORTALITY AND CLINICAL SIGNS OR OBSERVATIONS
In group 3, all animals had ruffled fur starting on day 4 or 5 of the pre-pairing period. Diarrhea was also observed in two males on day 9 of the pre-pairing period and in one female. The same female was found dead on day 11 of the pre-pairing period. Food consumption, relative food consumption and body weight were markedly reduced in males and females during the whole pre-pairing period. Therefore for ethical reasons, it was decided to terminate the group. At necropsy, dark red discoloration of lungs and thymus and bluish discoloration of uterus were observed in the female found dead.

In group 2, one female had ruffled fur on day 9 of the pre-pairing period. This was considered to be incidental since no other clinical sign was observed.


1.2 BODY TEMPERATURE
In group 3 body temperature on day 5 of the pre-pairing period was statistically significantly reduced in males (36.9 °C versus 37.6 °C in the control group) and in females (37.4 °C versus 38.4 °C).

Mean body temperature in group 2 was similar to the control values in males and females.


1.3 FOOD CONSUMPTION - MALES
Pre-pairing and After Pairing Periods:
In group 2, mean food consumption was reduced during the whole pre-pairing period ( 9.4% compared to the control). During the after pairing period, mean food consumption was similar to the control group during the first week of the period and lower at the end.


1.4 FOOD CONSUMPTION - FEMALES
Pre-pairing and Gestation Periods:
In group 2, mean food consumption was similar to the control during both pre-pairing and gestation periods.


1.5 BODY WEIGHTS - MALES
Pre-pairing, Pairing and After Pairing Periods:
In group 2, mean body weight and body weight gain were slightly lower during the pre-pairing period (overall body weight gain +8.3% versus +12.0%). During the pairing and after pairing periods, no differences in body weight gain were noted.


1.6 BODY WEIGHTS - FEMALES
Pre-pairing, Pairing and Gestation Periods:
In group 2, no relevant changes were observed in mean body weight and mean body weight gain during the pre-pairing, pairing and gestation periods.


1.7 CLINICAL LABORATORY INVESTIGATIONS
1.7.1 HEMATOLOGY
The assessment of the hematology data in group 2 did not reveal any test item-related effects in males and females.


1.7.2 CLINICAL BIOCHEMISTRY
The assessment of the clinical biochemistry parameters in group 2 did not reveal any test item-related effect in males and females.


1.8 REPRODUCTION DATA
1.8.1 MATING PERFORMANCE AND FERTILITY
No effects of the treatment with the test item on mating performance and fertility were noted. Mean precoital time was 3.0 and 2.0 days in groups 1 and 2, respectively and all females achieved a pregnancy.


1.8.2 IMPLANTATION RATE AND IMPLANTATION LOSS
In group 2, there was no indication of a test item-related effect in the number of corpora lutea, incidence of pre-implantation loss number of implantation sites and number of live embryos at any dose level.

Mean number of live embryos per dam in group 1 and 2 was 11.7 and 10.7, respectively.


1.9 TERMINAL FINDINGS
1.9.1 ORGAN WEIGHT DATA
In group 2, no changes in organ weight were noted.

1.9.2 MACROSCOPICAL FINDINGS
In group 2, dark red foci on the thymus were observed in two males and one female.


2 ADMINISTRATION IN THE DIET
2.1 MORTALITY AND CLINICAL SIGNS OR OBSERVATIONS
In group 7, all animals had ruffled fur starting on day 4 or 5 of the pre-pairing period. Mean food consumption, relative food consumption and body weight were markedly reduced, therefore it was decided for ethical reasons to terminate the group. At necropsy, no abnormal finding was observed.

In group 6, all animals had ruffled fur starting on day 9 of the pre-pairing period. Mean food consumption, relative food consumption and body weight were strongly decreased. Due to ethical reasons it was decided to terminate the group at the beginning of the pairing period.

No clinical signs were observed in group 5.


2.2 BODY TEMPERATURE
In group 7, mean body temperature recorded on day 5 of the pre-pairing period was statistically significantly reduced in males and females (37.2 °C versus 37.9 °C in males and 37.2 °C versus 38.8 °C in females.

In group 4, 5 and 6, mean body temperature on day 5 of the pre-pairing period was in males 37.9 °C, 37.8 °C and 37.3 °C, and in females 38.8 °C, 38.5 °C and 38.8 °C, respectively.


2.3 FOOD CONSUMPTION - MALES
Pre-pairing and After Pairing Periods:
In group 5, mean food consumption was reduced during the pre-pairing period (-7.0% compared to the control) and was slightly lower during the after pairing period (-3.1% compared to the control).


2.4 FOOD CONSUMPTION - FEMALES
Pre-pairing and Gestation Periods:
In group 5, mean food consumption was reduced at the beginning of the pre-pairing period ( 7.5% compared to the control) and slightly on days 11 - 14 (-4.0% compared to the control). During the gestation period, mean food consumption was reduced on days 7 - 14 (-13.2% compared to the control).


2.5 RELATIVE FOOD CONSUMPTION - MALES
Pre-pairing and After Pairing Periods:
In group 5, mean relative food consumption was reduced on days 1 - 4 of the pre-pairing period. Afterwards, mean relative food consumption remained slightly lower until the beginning of the after pairing period.


2.6 RELATIVE FOOD CONSUMPTION - FEMALES
Pre-pairing and Gestation Periods:
In group 5, mean relative food consumption was lower at the beginning of the pre-pairing period and during the second week of the gestation period.


2.7 BODY WEIGHTS - MALES
Pre-pairing, Pairing and After Pairing Periods:
In group 5, mean body weight and body weight gain were slightly lower during the pre-pairing (mean body weight gain +8.4% versus +10.6%) and pairing periods (mean body weight gain +2.9% versus +0.6%). These differences were not considered to be adverse. During the after pairing period no differences were noted (mean body weight gain +5.1% versus +5.3%).


2.8 BODY WEIGHTS - FEMALES
Pre-pairing, Pairing and Gestation Periods:
In group 5, no adverse effects were observed. Mean body weight and body weight gain were transiently lower on days 11 - 14 of the pre-pairing period. No effects were noted during the gestation (overall mean body weight gain +25.3% versus +24.6%).


2.9 CLINICAL LABORATORY INVESTIGATIONS
2.9.1 HEMATOLOGY
The assessment of the hematology data in group 5 did not reveal any test item-related effects in males and females.


2.9.2 CLINICAL BIOCHEMISTRY
The assessment of the clinical biochemistry parameters in group 5 did not reveal any test item-related effects in males and females.


2.10 REPRODUCTION DATA
2.10.1 MATING PERFORMANCE AND FERTILITY
Mean precoital time for groups 4 and 5 was 3.0 and 4.0 days respectively and all females achieved a pregnancy, therefore no effects of the test item were noted on the fertility.


2.10.2 IMPLANTATION RATE AND IMPLANTATION LOSS
No test item-related effects on the number of corpora lutea, incidence of pre-implantation loss number of implantation sites and number of live embryos were noted at any dose level.

Mean number of live embryos per dam in group 4 and 5 was 12.0 and 11.3, respectively.


2.11 TERMINAL FINDINGS
2.11.1 ORGAN WEIGHT DATA
In group 5, there was no indication of a test item-related effect on organ weights and ratios in males and females.


2.11.2 MACROSCOPICAL FINDINGS
In group 5, all males were noted to have several dark red foci on the thymus. No findings were observed in females.


3 BIONALYTICAL DATA

See IUCLID Section 7.5.1

Effect levels (P0)

Dose descriptor:

other: dose levels for main study

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

APPLICATION BY GAVAGE

REPRODUCTION DATA - SUMMARY OF PERFORMANCE

Group Dose (mg/kg)	1 (0)	2 (50)
Female numbers	10 - 12	13 - 15
Number of mated females	3	3
Number of pregnant females	3	3
Number of females with live embryos at termination*	3	3

*      Used for calculations

 

 

ADMINISTRATION IN THE DIET

REPRODUCTION DATA - SUMMARY OF PERFORMANCE

Group Dose (ppm)	4 (0)	5 (600)	6 (1800)
Female numbers	31 - 33	34 - 36	37 - 39
Number of mated females	3	3	1 (A)
Number of pregnant females	3	3
Number of females with live embryos at termination*	3	3

*      Used for calculations

(A)  Group 6 was terminated at the beginning of the pairing period.

 

 

TEST ITEM INTAKE - MALES

(See Figures on pp. 40 -41, Summary Tables on pp. 111 -112)

 

Pre-pairing Period

Generation	Concentration (ppm)	Mean achieved dose level (mg/kg bw/day)
P	0	0
	600	42.3
	1800	93.1
	6000 (days 1-8)	171.7

 

After Pairing Period

Generation	Concentration (ppm)	Mean achieved dose level (mg/kg bw/day)
P	0	0
	600	38.1

 

 

TEST ITEM INTAKE - FEMALES

(See Figures on pp. 42 -43, Summary Tables on pp. 113 -114)

 

Pre-pairing Period

Generation	Concentration (ppm)	Mean achieved dose level (mg/kg bw/day)
P	0	0
	600	49.7
	1800	126.2
	6000 (days 1-8)	254.3

 

Gestation Period

Generation	Concentration (ppm)	Mean achieved dose level (mg/kg bw/day)
P	0	0
	600	49.8

 

Applicant's summary and conclusion

Conclusions:

The purpose of this study was to select suitable dosages and appropriate administration method of the test item to be used in the subsequent reproduction/developmental toxicity screening test in the Han Wistar rat. The test item was therefore administered by gavage and by feeding. Additional biological assays and clinical pathology were also performed in order to evaluate further findings from previous studies.
The test item was administered orally by gavage once daily at dose levels of 50 and 150 mg/kg body weight/day and by feeding at dose levels of 600, 1800 and 6000 ppm.
Severe adverse effects occurred at dietary levels of 1800 and 6000 ppm and at the gavage dose level of 150 mg/kg/day. Consequently, these groups were terminated due to humane reasons. At the dietary level of 600 ppm and the gavage dose level of 50 mg/kg/day, similar findings were noted. There were no striking differences in findings when the test item was administered in the diet or by gavege. Based on these results dose levels of 10, 30 and 75 mg/kg bw/day were considered apriopriate for the subsequent Reproduction/Developmental Toxicity Screening Test in the Han Wistar Rat.

Executive summary:

The purpose of this study was to select suitable dosages and appropriate administration method to be used in the subsequent reproduction/developmental toxicity screening test in the Han Wistar rat.

APPLICATION BY GAVAGE

Three groups of 3 males and 3 females were treated by gavage with a similar supporting substance once daily. Males were treated over a 14-day pre-pairing period and during the pairing period and up to one day before necropsy (for at total of 29 days of treatment). Females were treated throughout the pre-pairing, pairing and gestation period up to day 13 post coitum (for a total of at least 29 days of treatment).

The following dose levels were applied:

Group 1:                        0 mg/kg body weight/day (control group)

Group 2:                      50 mg/kg body weight/day

Group 3:                    150 mg/kg body weight/day

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water).

The following results were obtained:

Mortality and General Tolerability

At 150 mg/kg/day, ruffled fur was observed in males and females starting on day 4 or 5 of the pre-pairing period. Diarrhea was also noted in two males and in one female on day 10 of the pre-pairing period. The same female was found dead on the following day. Food consumption and body weight gains were strongly reduced during the pre-pairing period. On day 1 of the pairing period, all males were in bad conditions therefore for ethical reasons it was decided to terminate the group. At necropsy, findings such as dark red discoloration of thymus and lungs, bluish discoloration of uterus and lungs not collapsed were observed in female found dead.

At 50 mg/kg/day, one female had ruffled fur on day 9 of the pre-pairing period. No other clinical signs were observed.

Body Temperature

At 150 mg/kg/day, mean body temperature on day 5 of the pre-pairing period was reduced in males and females.

At 50 mg/kg/day, mean body temperature recorded on day 5 of the pre-pairing period was similar to the control group.

Food Consumption and Body Weights

At 50 mg/kg/day, during the pre-pairing period mean food consumption and mean body weight were generally lower in males. In females, no effects on food consumption and body weights were observed.

Clinical Laboratory investigations

The assessment of hematology data and of clinical biochemistry parameters did not reveal any test item-related effects in males and females at 50 mg/kg/day.

Reproduction Data

At 50 mg/kg/day, relevant reproduction parameters such as number of corpora lutea, pre- and post-implantation loss and number of live embryos at termination were not affected.

Organ Weights

No changes in organ weight and ratios were observed.

Macroscopical Findings

At 50 mg/kg/day, dark red foci on thymus were observed in two males and in one female.

Bioanalytical Data

No inflammatory effects were observed. The increased values which occurred in single animals were within the normal range.

ADMINISTRATION IN THE DIET

The test item was administered in dietary levels of 0, 600, 1800 and 6000 ppm to four groups of 3 males and 3 females:

Group 4:                         0 ppm (control group)

Group 5:                     600 ppm

Group 6:                   1800 ppm

Group 7:                   6000 ppm

The following results were obtained:

Mortality and General Tolerability

At 6000 ppm, all animals showed ruffled fur starting on day 4 or 5 of the pre-pairing period. Food consumption, relative food consumption and body weight were strongly reduced. On day 11 of the pre-pairing period it was decided for ethical reasons to terminate the whole group.

At 1800 ppm, all animals had ruffled fur starting on day 5 or 9 of the pre-pairing period. Food consumption, relative food consumption and body weight were strongly decreased during the entire pre-pairing period. Based on these adverse effects it was decided to terminate the group.

At 600 ppm, no clinical signs were noted.

Body Temperature

At 6000 ppm, mean body temperature recorded on day 5 of the pre-pairing period was decreased in males and females.

At 600 ppm, body temperature recorded on day 5 of the pre-pairing period was similar to the control group.

Food Consumption and Body Weights

At 600 ppm, food consumption and relative food consumption were reduced at the beginning of the pre-pairing period in males and in females also during the second week of gestation but no statistical significance occurred. No adverse effects were observed in body weights of females and males.

Mean Test Item Intakes

The mean achieved dose levels of the test item in order of ascending dietary level were:

During the pre-pairing period:

Males                                                       42.3, 93.1 and 172 mg/kg body weight/day (only be­tween days 1 - 8)

Females                                                   49.7, 126.2 and 254 mg/kg body weight/day

During after pairing period:

Males                                                       38.1 mg/kg body weight/day

During the gestation period:

Females                                                   49.8 mg/kg body weight/day

Clinical Laboratory Investigations

The assessment of hematology data and clinical biochemistry parameters did not reveal any test item-related effects.

Reproduction Data

The relevant reproduction parameters such as number of corpora lutea, pre- and post-implantation loss and number of embryos were not affected.

Organ Weight

At 600 ppm, no changes were observed in organ weights and organ weight ratios in males and females.

Macroscopical Findings

At 600 ppm, all males had several dark red foci on the thymus.

Bioanalytical Data

No inflammatory effects were observed. The increased values which occurred in single animals were within the normal range.