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Repeated dose toxicity: oral

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Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 May 2012 to 15 Feb 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 May 2012 to 15 Feb 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: RCCHan™:WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst, Netherlands
- Age at study initiation: 10 weeks
- Weight at study initiation: males: 293 to 342 g, females: 204- 227 g
- Housing: Individually in Markrolon type-3 cages with wire mesh tops and sterilised standard softwood bedding with paper enrichment. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3. Values outside of this range occasionally occured, usually following room cleaning, which was considered not to have any influence on the study.
- Humidity (%): 30-70. Values outside of this range occasionally occured, usually following room cleaning, which was considered not to have any influence on the study.
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 07 June 2012 To: 20 July 2012
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG300
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared weekly. The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle was added (w/v). The mixtures were stirred using a magnetic stirrer and stored at room temperature (15- 25 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 20, 60 and 200 mg/mL/day. Dose volume: 5mL/kg bodyweight.
- Lot/batch no. (if required): BCBF5743V
Details on mating procedure:
Mating, gestation and lactation
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if:
-      The daily vaginal smear was positive, or
-      A copulation plug was observed.

The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

If a female did not mate during the 14-d period, a second pairing of this female with a male in the same group, which had already mated successfully, was considered. If mating was not recorded during the additional pairing period of a maximum of 14 d, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for infertility.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulations were analysed using a GC-FID method.
Duration of treatment / exposure:
Males: up to 41 d
Females: up to 53 d
Frequency of treatment:
Once daily
Details on study schedule:
Study Sequence
Acclimatisation: 7 d for males and females
First test item administration: Day 1 of pre-pairing for males and females
Pre-pairing: 14 d for males and females
Blood sampling: end of pre-pairing for males and females
Pairing: 14 d maximum for males and females
Gestation: approximately 21 d for females only
Treatment ends: on day of dacrifice for males, on day 3 post partum for females
Necropsy: after treatement of at least 28 d, when no longer needed for assessment of reproductive effects for males. On day 4 post partum (pups on day 4 post partum) for females.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1 (control)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
11/ sex/ dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected based on a previous non-GLP dose range-finsing study in Wistar rats
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatisation and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioural abnormalities in nesting and nursing.

Once prior to the first administration of the test item and weekly thereafter, detailed clinical observations were performed outside the home cage. In females, it was performed on days 0, 6, 13 and 20 of the gestation period. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behaviour were also reported.

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

FOOD CONSUMPTION: Yes. No food consumption was recorded during the pairing period.
-Males: weekly during pre-pairing and after pairing periods
- Females: pre-pairing period days 1-8 and 8-14; gestation days 0-7, 7-14 and 14-21 post coitum, and days 1-4 post partum.

HAEMATOLOGY: Yes
Blood samples were obtained at the end of the pre-pairing period from 5 males and 5 females from each group. Blood samples were drawn retro-orbitally from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 h 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.

The following haematology parameters were determined:

Complete blood cell count
Erythrocyte count, haemoglobin, haematocrit, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, leukocyte count (total), mean corpuscular volume, red cell volume distribution width, mean corpouscular haemoglobin, 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 phosphate, gamma-glutamyl-transferase, bile acids, sodium, potassium, chloride, calcium, phosphorous, protein (total), albumin, globulin, albumin/ globulin ratio

OTHER:
FUNCTIONAL OBSERVATION BATTERY (FOB)
At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum), relevant parameters from modified Irwin screen test was performed with 5 P generation males and 5 P generation females from each group in place of the usual weekly behavioural observation. This FOB assessment was conducted following the daily dose administration.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151. Activity of the animals (based on beam count) was recorded for 10 minute intervals over a period of 60 min. These data and the total activity over 60 min were reported.
Litter observations:
STANDARDISATION OF LITTERS
The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (wihtout identification) on days 0 (if possible), 1 and 4 post partum.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All survivng animals were sacrificed after treatment of at least 28 d, when no longer needed for the assessment of reproductive effects
- Maternal animals: All surviving animals were sacrificed on day 4 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

GROSS NECROPSY
All animals sacrificed or found dead were weighted and subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4 % formaldehyde solution.

At the scheduled sacrifice, all animals were sacrificed by an injection of sodium pentobarbital or were sacrificed by CO2 asphyxiation. All P generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

For the parent animals, special attention was directed at the organs of the reproductive system.
The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulphide to visualise possible haemorrhagic areas of implantation sites.

HISTOPATHOLOGY / ORGAN WEIGHTS
At the scheduled sacrifice, the testes and epididymides of all parental males were weighted separately.

In addition, from 5 males and 5 females of each group, sacrificed at the end of the study, the following organs 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.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring: All surviving animals were sacrificed on day 4 post partum.

GROSS NECROPSY
Dead pups, except those excessively cannibalised, were examined macroscopically.

All pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
1. Means and standard deviations of various data were calculated.
2. The Dunnett-test (many to one t-test) based on the 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.
3. 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.
4. Fisher’s exact-test was applied if the variables could be dichotomised without loss of information.
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY
Viability/ mortaility
One male treated with 1000 mg/kg/day was found dead spontaneously on day 12 of the pre-pairing period.

One female treated with 1000 mg/kg/day of the test item was found spontaneously dead on day 11 of the pre-pairing and another female of this group was found dead on day 22 of the gestation period. Two further females of this group were found dead on day 2 of their lactation period.

All these deaths deemed to be caused by aspiration during gavage procedures of massive amount test item and it could be considered as the cause of death, not related to systemic toxicity of the test item. All other animals of this group survived until the scheduled necropsy.

All animals treated with 100 mg/kg/day or 300 mg/kg/day of the test item survived until the scheduled necropsy.

Daily clinical signs or observations
A kinked tail apex with scabs was seen in 1 control male from the treatment day 6 onwards, this was black coloured, additionally, from day 9 onwards. During the pairing period moderate necrosis of this apex was seen additionally. One male treated with 100 mg/kg/day of the test item had slight hair loss on the right shoulder from day 8 and another male of this group had slight breathing noises on one day. One male treated with 1000 mg/kg/day had slight breathing noises on up to 9 days of the pre-pairing period and also on 3 days on the pairing period.

Slight breathing noises and slight hair loss on the head were noted in 1 female treated with 300 mg/kg/day of the test item on single days of the pre-pairing period. Slight breathing noises were noted also in a few females treated with 1000 mg/kg/day on some single days of the pre-pairing and pairing period and slight hair loss on the neck in 1 female of this group. Slightly to moderately ruffled fur was seen in a few females of this group on 3 days. One female neglected its litter and no milk was found in the stomach of the pups during the first litter check. The reason for this is unclear and it was considered to be an incidental finding.

One female treated with 1000 mg/kg/day had a mass on the neck in the gestation period. The mass was still seen during the lactation period. This finding was considered to be incidental.

All these possible findings were considered to be incidental and the slight breathing noises could be possibly treatment-related but not test item-related.

Findings at detailed weekly clinical observations
No toxicological relevant test item-related clinical signs were noted during the weekly observations.

One female treated with 1000 mg/kg/day showed ruffled fur on day 13 and another female of this dose group had a mass on the neck on day 20 post coitum.

FOOD CONSUMPTION, BODY WEIGHT AND WEIGHT GAIN
PRE-PAIRING PERIOD
There were no differences of toxicological relevance between the mean food consumption of test item-related or control males. The mean relative food consumption of test item treated groups was similar to this of the controls.

PRE-PAIRING, GESTATION AND LACTATION PERIODS
Females treated with 1000 mg/kg/day showed a tendency of a slightly decreased mean absolute and relative food consumption. This slight decrease was considered to be not adverse because it is a slight effect without statistical significance and no dose response relationship could be observed.

PRE-PAIRING AND PAIRING PERIODS
There were no differences of toxicological relevance between the mean body weights of test item-treated or control males. The mean body weight gain of all groups compared favourably.

PRE-PAIRING, PAIRING, GESTATION AND LACTATION PERIODS
On some days of the gestation and in the lactation period, mean body weight was slightly decreased (p <0.05) in the 1000 mg/kg/day treatment group when compared to the control animals. This was considered to be not adverse because it was seen on some single days only. No test item-related effects on body weights and body weight gain of females were observed during the pre-pairing and pairing period.

No test item-related effect on body weights and body weight gain of females treated with 100 mg/kg/day or 300 mg/kg/day of the test item were observed during the study.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Eleven, 10, 11 and 10 females in the treatment groups 0, 100, 300 and 1000 mg/kg/day respectively, were mated within the first or second pairing.

The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 3.9, 3.7, 2.4 and 3.2 days in treatment groups 0, 100, 300 and 1000 mg/kg/day respectively. The median precoital time was 2, 3, 2 and 3 days in ascending dose level. For the one control female that mated during the second pairing period, precoital time was 1 d.

The fertility index was 100.0 %, 90.9 %, 100.0 %, 100.0 % and the conception rate showed exactly the same values in the groups treated with 0 mg/kg/day, 100 mg/kg/day, 300 mg/kg/day or 1000 mg/kg/day, respectively.

ORGAN WEIGHTS
No test –item-related changes in the mean organ weights, organ to body weight ratios and organ to brain weight ratios were noted in males and females when compared with the control animals.

GROSS PATHOLOGY
There were no gross lesions that could be attributed to treatment with the test item in sacrificed animals. Although the liver enlargement was recorded in a few males including control group animals, there was no histological correlate. All other gross lesions recorded were considered to be within the range of normal background alterations.

HISTOPATHOLOGY: NON-NEOPLASTIC
The respiratory disorder consisted of necrosis and inflammatory cell infiltration of the trachea, congestion, alveolar edema and alveolar macrophaes of the lung were recorded in dead animals. Major microscopic findings with macroscopic findings in each animal were described as follows:
FINDINGS IN DEAD ANIMALS
Animal No. 34
Trachea: moderate mucosal necrosis and slight inflammatory cell infiltration were recorded and acute reactive and inflammatory changes were considered.
Lung: reactive change consisting of slight congestion and slight alveolar macrophages was recorded. (Macroscopic findings: discolouration, dark red)
Thymus: multifocal slight haemorrhage as lesion during agonal period. (Macroscopic findings: focus/ foci, dark red)

Animal No. 83
Trachea: moderate mucosal necrosis and moderate inflammatory cell infiltration were recorded and acute reactive and inflammatory changes were considered.
Lung: reactive change consisting of moderate congestion, slight alveolar edema and slight alveolar macrophages was recorded. (Macroscopic findings: discolouration, dark red).

Animal No. 87
Trachea: ,oderate mucosal necrosis and moderate inflammatory cell infiltration were recorded and acute reactive and inflammatory changes were considered.
Lung: reactive change consisting of minimal congestion and slight alveolar macrophages was recorded. (Macroscopic findings: discolouration, dark red)
Thymus: multi focal slight haemorrhage as lesion during agonal period. (Macroscopic findings: gelatinous, focus/ foci, dark red)

Animal No.88
Trachea: moderate inflammatory cell infiltration was recorded and acute reactive and inflammatory changes were considered.
Lung: reactive change consisting of moderate congestion, slight alveolar edema and minimal alveolar macrophages was recorded. (Macroscopic findings: discolouration, dark red)

FINDINGS IN SACRIFIED ANIMALS
All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

OTHER FINDINGS (PARENTAL ANIMALS)
CORPORA LUTEA COUNT
Mean number of corpora lutea per dam (determined at necropsy) was similar in all groups (14.9, 13.4, 13.8 and 14.2 in order of ascending dose level) and gave no indication of a test item-related effect.

DURATION OF GESTATION
The mean duration of gestation was unaffected by exposure to the test item. Mean duration of gestation was 21.4, 21.8, 21.5 and 21.6 d, in order of ascending dose level.

IMPLANTATION RATE AND POST-IMPLANTATION LOSS
No test item-related effects were noted on implantation rate and implantation loss.

The total post-implantation loss was significantly decreased (p< 0.05) in females treated with 1000 mg/kg/day of the test item when compared with the controls, which was considered to be incidental.

The mean numbers of implantations per litter were 13.6, 11.4, 13.1 and 13.1 in order of ascending dose level. The mean incidence of post-implantation loss as a percentage of total implantations was 16.0, 17.5, 13.9 and 8.5 %, in order of ascending dose level.

LITTER SIZE AT FIRST LITTER CHECK
No effects were noted on litter size at first litter check.

In dose groups 0, 100, 300 and 1000 mg/kg/ day the birth index was 84.0, 83.5, 86.1 and 91.5% and mean litter size at first litter check was 11.5, 9.6, 11.3 and 12.0, respectively.

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM
No effects on the postnatal loss between day 0 and 4 post partum were noted.

Mean postnatal loss was 1.6, 1.2, 1.6 and 9.6% in dose groups 0, 100, 300 and 1000 mg/kg/ day, respectively. Correspondingly, in dose group 1000 mg/kg/day, the viability index was statistically significantly decreased (90.4%). In does groups 0, 100 and 300 mg/kg/day, the viability index was 98.4, 98.8 and 98.4%.

The cause of the slightly higher postnatal loss in dose group 1000 mg/kg/day was the loss of 7 pups on day 2 and 3 post partum for dam no. 84. For this dam already 6 dead pups at first litter check were noted. This isolated occurrence was considered to be incidental.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
CLINICAL SIGNS (OFFSPRING)
No abnormal findings were noted at first litter check or during the first 4 days post partum.

BODY WEIGHT (OFFSPRING)
Mean pup weights on day 1 and day 4 post partum were unaffected by treatment of their dams with the test item.

The mean difference in pup weight in the 1000 mg/kg/day treatment group, was slightly decreased (P< 0.05) when compared male and female pups of this group with the control group. Since the difference was not statistically significant, it was considered to be not test item-related.

GROSS PATHOLOGY (OFFSPRING)
No test item-related macroscopic findings were noted in pups at necropsy.
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test-item related findings were noted at first litter check and during lactation in pups at any dose level
Critical effects observed:
no
Reproductive effects observed:
no

Summary of performance

P animals breeding for F1 litters

Group

(mg/kg/day)

1

(0)

2

(100)

3

(300)

4

(1000)

Female numbers

45 - 55

56 – 66

67 – 77

78 – 88

Number of females paired (A)

11

11

11

10

Number of females mated

11

10

11

10

Number of females, which died before the scheduled necropsy (B)

0

0

0

4

Number of females not pregnant (C)

0

1

0

0

Number of females which reared their pups until day 4post partum

11

9

11

7

(A) Female no. 88 died spontaneously during pregnancy

(B)Females died spontaneously, no.88 during pre-pairing, 86 during gestation, 83/87 during lactation

(C)Female no. 63 was not pregnant

Conclusions:
The reproductive toxicity via the oral route of the test item was assessed according to OECD Guideline 422 at dose levels of 100, 300 and 1000 mg/kg/day. Based on the results of the study, a NOAEL for general toxicity in males and females was considered to be 1000 mg/kg/day, the highest dose level tested.
Executive summary:

A 28 day repeat dose oral toxicity study combined with a reproductive/ developmental toxicity screening test was performed in the rat in accordance with OECD Guideline 422. The test item was administered by gavage to 3 groups, each of 11 male and 11 female RCCHan™:WIST strain rats for up to 8 weeks (including a 2 week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. There were no treatment related effects observed on mating, fertility or gestation length at any dose level. The offspring litter size, sex ratio, viability, growth and development were al comparable to controls and no adverse effects were noted. Since no treatment-related effects were observed for reproduction, a NOAEL was considered to be 1000 mg/kg bw/day. Furthermore, the study showed that the administration of the test item over a period of 28 days did not results in any toxicologically significant effects and hence the NOAEL for reproduction/ developmental toxicity was considered to be 1000 mg/kg bw/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Cis-hex-3-en-ol, leaf alcohol
- Physical state: liquid
- Analytical purity: 98.59 %
- Lot/batch No.: 1Z50650
- Expiration date of the lot/batch: 06 June 2015
- Storage condition of test material: at room temperature (15- 25 °C) away from direct sunlight.

Test animals

Species:
rat
Strain:
other: RCCHan™:WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst, Netherlands
- Age at study initiation: 10 weeks
- Weight at study initiation: males: 293 to 342 g, females: 204- 227 g
- Housing: Individually in Markrolon type-3 cages with wire mesh tops and sterilised standard softwood bedding with paper enrichment. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3. Values outside of this range occasionally occured, usually following room cleaning, which was considered not to have any influence on the study.
- Humidity (%): 30-70. Values outside of this range occasionally occured, usually following room cleaning, which was considered not to have any influence on the study.
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 07 June 2012 To: 20 July 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG300
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared weekly. The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle was added (w/v). The mixtures were stirred using a magnetic stirrer and stored at room temperature (15- 25 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 20, 60 and 200 mg/mL/day. Dose volume: 5mL/kg bodyweight.
- Lot/batch no. (if required): BCBF5743V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulations were analysed using a GC-FID method.
Duration of treatment / exposure:
Males: up to 41 d
Females: up to 53 d
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1(Control group)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
11/ sex/ dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected based on a previous non-GLP dose range-finsing study in Wistar rats

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatisation and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioural abnormalities in nesting and nursing.

Once prior to the first administration of the test item and weekly thereafter, detailed clinical observations were performed outside the home cage. In females, it was performed on days 0, 6, 13 and 20 of the gestation period. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behaviour were also reported.

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

FOOD CONSUMPTION: Yes. No food consumption was recorded during the pairing period.
-Males: weekly during pre-pairing and after pairing periods
- Females: pre-pairing period days 1-8 and 8-14; gestation days 0-7, 7-14 and 14-21 post coitum, and days 1-4 post partum.

HAEMATOLOGY: Yes
Blood samples were obtained at the end of the pre-pairing period from 5 males and 5 females from each group. Blood samples were drawn retro-orbitally from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 h 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.

The following haematology parameters were determined:

Complete blood cell count
Erythrocyte count, haemoglobin, haematocrit, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, leukocyte count (total), mean corpuscular volume, red cell volume distribution width, mean corpouscular haemoglobin, 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 phosphate, gamma-glutamyl-transferase, bile acids, sodium, potassium, chloride, calcium, phosphorous, protein (total), albumin, globulin, albumin/ globulin ratio

OTHER:
PUP DATA
The liiters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually on days 0 (if possible), 1 and 4 post partum.

FUNCTIONAL OBSERVATION BATTERY (FOB)
At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum), relevant parameters from modified Irwin screen test was performed with 5 P generation males and 5 P generation females from each group in place of the usual weekly behavioural observation. This FOB assessment was conducted following the daily dose administration.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151. Activity of the animals (based on beam count) was recorded for 10 minute intervals over a period of 60 min. These data and the total activity over 60 min were reported.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Males were sacrificed after treatment of at least 28 d, when no longer needed for the assessment of reproductive effects. Dams and pups were sacrificed on day 4 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

All animals sacrificed or found dead were weighted and subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4 % formaldehyde solution. At the scheduled sacrifice, all animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated. Dead pups, except those excessively cannibalised, were examined macroscopically.

All parent animals were examined macroscopically for any structural changes at the scheduled necropsy or during the study if death occurred. Also pups were examined macroscopically for any structural changes at the scheduled necropsy and if possible also if death occured.

For the parent animals, special attention was directed at the organs of the reproductive system.

The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulphide to visualise possible haemorrhagic areas of implantation sites.
Other examinations:
Organ weights
At the scheduled sacrifice, the testes and epididymides of all parental males were weighted separately.

In addition, from 5 males and 5 females of each group, sacrificed at the end of the study, the following organs 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.
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
1. Means and standard deviations of various data were calculated.
2. The Dunnett-test (many to one t-test) based on the 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.
3. 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.
4. Fisher’s exact-test was applied if the variables could be dichotomised without loss of information.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Viability/ mortaility
One male treated with 1000 mg/kg/day was found dead spontaneously on day 12 of the pre-pairing period.

One female treated with 1000 mg/kg/day of the test item was found spontaneously dead on day 11 of the pre-pairing and another female of this group was found dead on day 22 of the gestation period. Two further females of this group were found dead on day 2 of their lactation period.

All these deaths deemed to be caused by aspiration during gavage procedures of massive amount test item and it could be considered as the cause of death, not related to systemic toxicity of the test item. All other animals of this group survived until the scheduled necropsy.

All animals treated with 100 mg/kg/day or 300 mg/kg/day of the test item survived until the scheduled necropsy.

Daily clinical signs or observations
A kinked tail apex with scabs was seen in 1 control male from the treatment day 6 onwards, this was black coloured, additionally, from day 9 onwards. During the pairing period moderate necrosis of this apex was seen additionally. One male treated with 100 mg/kg/day of the test item had slight hair loss on the right shoulder from day 8 and another male of this group had slight breathing noises on one day. One male treated with 1000 mg/kg/day had slight breathing noises on up to 9 days of the pre-pairing period and also on 3 days on the pairing period.

Slight breathing noises and slight hair loss on the head were noted in 1 female treated with 300 mg/kg/day of the test item on single days of the pre-pairing period. Slight breathing noises were noted also in a few females treated with 1000 mg/kg/day on some single days of the pre-pairing and pairing period and slight hair loss on the neck in 1 female of this group. Slightly to moderately ruffled fur was seen in a few females of this dose group on 3 days in the lactation period and 1 day during gestation. In females slgiht breathing noises were noted also during a few days of the gestation and lactation period. One female neglected its litter and no milk was found in the stomach of the pups during the first litter check. The reason for this is unclear and it was considered to be an incidental finding.

One female treated with 1000 mg/kg/day had a mass on the neck in the gestation period. The mass was still seen during the lactation period. This finding was considered to be incidental.

All these possible findings were considered to be incidental and the slight breathing noises could be possibly treatment-related but not test item-related.

Findings at detailed weekly clinical observations
No toxicological relevant test item-related clinical signs were noted during the weekly observations.

One female treated with 1000 mg/kg/day showed ruffled fur on day 13 and another female of this dose group had a mass on the neck on day 20 post coitum.

FOOD CONSUMPTION, BODY WEIGHT AND WEIGHT GAIN
PRE-PAIRING PERIOD
There were no differences of toxicological relevance between the mean food consumption of test item-related or control males. The mean relative food consumption of test item treated groups was similar to this of the controls.

PRE-PAIRING, GESTATION AND LACTATION PERIODS
Females treated with 1000 mg/kg/day showed a tendency of a slightly decreased mean absolute and relative food consumption. This slight decrease was considered to be not adverse because it is a slight effect without statistical significance and no dose response relationship could be observed.

PRE-PAIRING AND PAIRING PERIODS
There were no differences of toxicological relevance between the mean body weights of test item-treated or control males. The mean body weight gain of all groups compared favourably.

PRE-PAIRING, PAIRING, GESTATION AND LACTATION PERIODS
On some days of the gestation and in the lactation period, mean body weight was slightly decreased (p <0.05) in the 1000 mg/kg/day treatment group when compared to the control animals. This was considered to be not adverse because it was seen on some single days only. No test item-related effects on body weights and body weight gain of females were observed during the pre-pairing and pairing period.

No test item-related effect on body weights and body weight gain of females treated with 100 mg/kg/day or 300 mg/kg/day of the test item were observed during the study.

HAEMATOLOGY
MALES
The assessment of the haematology data did not reveal any test item-related effects.

The differences of haematological parameters of test item treated males compared with the control animals show no dose response relationship. Therefore these changes were considered to be not test item-related.

FEMALES
The assessment of the haematology data did not reveal any test item-related effects.

The slightly decreased (p< 0.05) mean corpuscular haemoglobin concentration in females treated with 300 mg/kg/day or 1000 mg/kg/day of the test item compared to controls are within the range of the historical reference data. The mean value of relative monocytes was slightly decreased (P< 0.05) only in females treated with 300 mg/kg/day when compared with controls. Therefore no dose response relationship could be observed. The differences of these both parameters were therefore considered to be not test item-related.

BIOCHEMISTRY
MALES
No test item-related differences of biochemistry were noted when compared with control animals at the end of the pre-pairing period.

The slight increase (P< 0.01) of the mean chloride content in males treated with 300 mg/kg/day or 1000 mg/kg/day of the test item was within the range of the historical reference data and therefore considered to be not test item-related.

FEMALES
No test item-related differences in parameters of biochemistry were noted when compared with control animals at the end of the pre-pairing period.

ORGAN WEIGHTS
No test –item-related changes in the mean organ weights, organ to body weight ratios and organ to brain weight ratios were noted in males and females when compared with the control animals.

GROSS PATHOLOGY
There were no gross lesions that could be attributed to treatment with the test item in sacrificed animals. Although the liver enlargement was recorded in a few males including control group animals, there was no histological correlate. All other gross lesions recorded were considered to be within the range of normal background alterations.

HISTOPATHOLOGY: NON-NEOPLASTIC
The respiratory disorder consisted of necrosis and inflammatory cell infiltration of the trachea, congestion, alveolar edema and alveolar macrophaes of the lung were recorded in dead animals. Major microscopic findings with macroscopic findings in each animal were described as follows:
FINDINGS IN DEAD ANIMALS
Animal No. 34
Trachea: moderate mucosal necrosis and slight inflammatory cell infiltration were recorded and acute reactive and inflammatory changes were considered.
Lung: reactive change consisting of slight congestion and slight alveolar macrophages was recorded. (Macroscopic findings: discolouration, dark red)
Thymus: multifocal slight haemorrhage as lesion during agonal period. (Macroscopic findings: focus/ foci, dark red)

Animal No. 83
Trachea: moderate mucosal necrosis and moderate inflammatory cell infiltration were recorded and acute reactive and inflammatory changes were considered.
Lung: reactive change consisting of moderate congestion, slight alveolar edema and slight alveolar macrophages was recorded. (Macroscopic findings: discolouration, dark red).

Animal No. 87
Trachea: ,oderate mucosal necrosis and moderate inflammatory cell infiltration were recorded and acute reactive and inflammatory changes were considered.
Lung: reactive change consisting of minimal congestion and slight alveolar macrophages was recorded. (Macroscopic findings: discolouration, dark red)
Thymus: multi focal slight haemorrhage as lesion during agonal period. (Macroscopic findings: gelatinous, focus/ foci, dark red)

Animal No.88
Trachea: moderate inflammatory cell infiltration was recorded and acute reactive and inflammatory changes were considered.
Lung: reactive change consisting of moderate congestion, slight alveolar edema and minimal alveolar macrophages was recorded. (Macroscopic findings: discolouration, dark red)

FINDINGS IN SACRIFIED ANIMALS
All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

OTHER FINDINGS
FUNCTIONAL OBSERVATIONAL BATTERY
None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect. Mean values of grip strength (fore- and hind paws) of males and females gave no indication of test item-related effects. The mean body temperature of test item treated males and females were similar to this of control animals.

LOCOMOTOR ACTIVITY
The mean locomotor activity of females treated with 1000 mg/kg/day was slightly decreased (p< 0.05 at 20-30 min interval) when compared with controls during the lactation period. No test item-related effects on locomotor activity of males and females of the other dose groups were noted.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects at 1000 mg/kg bw/day in any of the parameters assessed

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Reproduction and Breeding Data

Summary of performance

P animals breeding for F1 Litters

Group

(mg/kg/day)

1

(0)

2

(100)

3

(300)

4

(1000)

Female numbers

45 - 55

56 - 66

67 – 77

78 – 88

Number of females paired (A)

11

11

11

10

Number of females mated

11

11

11

10

Number of females, which dies before the scheduled necropsy (B)

0

0

0

4

Number of females not pregnant (C)

0

1

0

0

Number of females which reared their pups until day 4 post partum

11

9

11

7

(A) Female no. 88 died spontaneously during preparing

(B) Females died spontaneously, no. 88 during pre-pairing, 86 during gestation, 83/87 during lactation

(C) Female no. 63 was not pregnant

Mating Performance and Fertility

Eleven, 10, 11 and 10 females in dose groups 0, 100, 300 and 1000 mg/kg/ day, respectively, were mated within the first or second pairing period.

The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 3.9, 3.7, 2.4 and 3.2 days in dose groups 0, 100, 300 and 1000 mg/kg/ day, respectively. The median precoital time was 2, 3, 2 and 3 days in order of ascending dose level. For the 1 control female that mated during the second pairing period, precoital time was 1 day.

The fertility index was 100.0%, 90.9%, 100.0%, 100.0% and the conception rate showed exactly the same values in the groups treated with 0 mg/kg/day, 100 mg/kg/day, 300 mg/kg/day or 1000 mg/kg/day, respectively.

Corpora Lutea Count

Mean number of corpora lutea per dam (determined at necropsy) was similar in all groups (14.9, 13.4, 13.8 and 14.2 in order of ascending dose level) and gave no indication of a test item-related effect.

Duration of Gestation

The mean duration of gestation was unaffected by exposure to the test item. Mean duration of gestation was 21.4, 21.8, 21.5 and 21.6 days, in order of ascending dose level.

Implantation Rate and Post-implantation Loss

No test item-related effects were noted on implantation rate and implantation loss.

The total post-implantation loss was significantly decreased (p<0.05) in females treated with 1000 mg/kg/day of the test item when compared with the controls, which was considered to be incidental. The mean numbers of implantations per litter were 13.6, 11.4, 13.1 and 13.1 in order of ascending dose level. The mean incidence of post-implantation loss as a percentage of total implantations was 16.0, 17.5, 13.9 and 8.5%, in order of ascending dose level.

Litter Size at First Litter Check

No effects were noted on litter size at first litter check.

In dose groups 0, 100, 300 and 1000 mg/kg/ day the birth index was 84.0, 83.5, 86.1 and 91.5% and mean litter size at first litter check was 11.5, 9.6, 11.3 and 12.0, respectively.

Postnatal Loss Days 0 - 4 Post Partum

No effects on the postnatal loss between day 0 and 4 post partum were noted.

Mean postnatal loss was 1.6, 1.2, 1.6 and 9.6% in dose groups 0, 100, 300 and 1000 mg/kg/ day, respectively. Correspondingly, in dose group 1000 mg/kg/day, the viability index was statistically significantly decreased (90.4%). In does groups 0, 100 and 300 mg/kg/day, the viability index was 98.4, 98.8 and 98.4%.

The cause of the slightly higher postnatal loss in dose group 1000 mg/kg/day was the loss of 7 pups on day 2 and 3 post partum for dam no. 84. For this dam already 6 dead pups at first litter check were noted. This isolated occurrence was considered to be incidental.

Litter Data - F1 Pups

External Examination at First Litter Check and during Lactation

No abnormal findings were noted at first litter check or during the first 4 days post partum.

Sex Ratios

Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item.

Body Weights to Day 4 Post Partum

Mean pup weights on day 1 and day 4 post partum were unaffected by treatment of their dams with the test item.

The mean difference in pup weight in dose group 1000 mg/kg/day, was slightly decreased (p<0.05) when compared male and female pups of this group with the control group. Since the difference of the mean absolute body weights of pups on days 1 and 4 post partum was not statistically significant, it was considered to be not test item related.

Macroscopical Findings

No test item-related macroscopic findings were noted in pups at necropsy.

Applicant's summary and conclusion

Conclusions:
The repeated dose toxicity via the oral route of the test item was assessed according to OECD Guideline 422 at dose levels of 100, 300 and 1000 mg/kg/day. Based on the results of the study, a NOAEL for general toxicity in males and females was considered to be 1000 mg/kg/day, the highest dose level tested.