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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 August 2012 to 16 April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, equivalent to a valid guidelines and the study was conducted under GLP conditions.
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 August 2012 to 16 April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, equivalent to a valid guidelines and the study was conducted under GLP conditions.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst, The Netherlands
- Weight at study initiation: 291 - 367 g (males); 189 - 239 g (females)
- Housing: individually in Makrolon type-3 cages with wire mesh tops
- Diet: Pelleted standard Harlan Teklad 2914C ad libitum
- Water: tap water ad libitum
- Acclimatisation 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: 6th September 2012: To: 15th October 2012
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was weighed into a glass beaker and the vehicle added. The mixtures were stirred using a magnetic stirrer and stored at room temperature.

VEHICLE
- Lot/batch no. (if required): BCBG8285V
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days maximum
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no (after two unsuccessful matings, the female was sacrificed and the reproductive organs examined histopathologically)
- After successful mating each pregnant female was caged (how): Individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulations were analysed by GC-FID.

Concentration, homogeneity and stability were determined in samples taken after experimental start (see Table 1 in 'Any other information on materials and methods incl. tables').

Concentration and homogeneity were further determined (see Table 2 in 'Any other information on materials and methods incl. tables').
Duration of treatment / exposure:
males: 4 weeks; females: 7 weeks
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle control (Group 1)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
700 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
11
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected on the basis of a non-GLP range-finding study (see details in repeat dose enpoint for OECD 422 study)
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
Observations: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity. Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes or bizarre behaviour were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly (males); pre-mating period days 1-8 and 8-13 (females)

WATER CONSUMPTION: No

OTHER: neurobehavioural examination, clinical chemistry, and haematology were all examined and reported in the corresponding repeated dose toxicity summary.
Sperm parameters (parental animals):
- Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure. If test material-related morphologic changes were detected in organs of high-dose animals, the same organs from mid-dose and low-dose animals were examined to establish a no-effect level if possible. Microscopic examination of the reproductive organs of all infertile males was made.
Litter observations:
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 (without identification) on days 0 (if possible) 1 and 4 post-partum.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving males were sacrificed after at least 28 days treatment with the test material (when the males were no longer needed for the assessment of reproductive effects)
- Maternal animals: All surviving females were sacrificed on day 4 post-partum.

GROSS NECROPSY / ORGAN WEIGHTS
- Gross necropsy consisted of the testes and epididymides of all parental males were weighed separately. In addition, the following organs were selected from five males and five females from each group and weighed: adrenals, brain, heart, kidneys, liver, thymus and spleen.

HISTOPATHOLOGY
The following tissues were examined histologically: prostrate, seminal vesicles with coagulating gland, testes and epididymides (males); ovaries 9females); gross lesions, brain, spinal cord, heart, thymus, thyroids and parathyroids, small and large intestines, stomach, liver, kidneys, adrenals, spleen, trachea and lungs, uterus, urinary bladder, lymph nodes, peripheral nerve and bone marrow (males and females).
Postmortem examinations (offspring):
Pups were observed for any gross anomalies.
Statistics:
The following statistical methods were used to analyse food consumption, body weights and reproduction data:
- Mean and standard deviations were calculated.
- The Dunnett-test based on 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 was applied instead of the Dunnett test when the could not be assumed to follow a normal distribution.
- Fisher's exact test was applied if the variables could be dichotomised without loss of information.
Reproductive indices:
Corpora lutea count
Duration of gestation
Implantation rate and post-implantation loss
Litter size
Offspring viability indices:
Sex ratios
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, non-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:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One control male was found spontaneously dead on day five and one 200 mg/kg bw/day male was found spontaneously dead on day 11; both deaths were considered to be due to an incidental influx of dosing solution into the respiratory tract. Two 700 mg/kg bw/day males were found spontaneously dead on days five and six of the pre-mating period.

Two 200 mg/kg bw/day females were found spontaneously dead on days 23 and 24 of the gestation period; the death of one of them was considered to be due to incidental influx of dosing solution into the respiratory tract. Three 700 mg/kg bw/day females were found spontaneously dead on days five, six and seven of the pre-mating period, one female of this dose group was sacrificed in extremis on day two of the pre-mating period and another female of this group was sacrificed in extremis on day 18 of the gestation period.

Apart from the deaths considered to be due to incidental influx of dosing solution into the respiratory tract, the death of one 200 mg/kg bw/day female was considered to probably be pregnancy toxemia but this was not considered to be directly attributable to the test material. In the remainder of the animals, the worsened general condition which was caused by the effects on the forestomach and kidneys and the continual stressful condition attributable to treatment with the test material were considered to be the cause of death.

No clinical signs were noted in control and 50 mg/kg bw/day males during the pre-mating period. Slight breathing noises were noted in a few 200 and 700 mg/kg bw/day males on one or two days of the pre-mating period. During the mating period, slightly soft faeces were noted in a few control males on one day. Slight breathing noises were noted in a few 50 mg/kg bw/day males on two days and in some 200 and 700 mg/kg bw/day males on some days of the mating period.

No clinical signs were noted in control, 50 and 200 mg/kg bw/day females during the pre-mating period. Slight breathing noises were noted in 700 mg/kg bw/day females on some days of the pre-mating period. Prostration and hunched posture were noted in some 700 mg/kg bw/day females on single days. Additionally, slightly ruffled fur and visible slight weight loss were seen in 700 mg/kg bw/day females on some days, slight convulsion was noted in one female on on day and slightly decreased activity was noted in a few females on one day of the pre-mating period.

During the mating period, no clinical signs were noted in control, 50 and 200 mg/kg bw/day females. Slightly decreased activity, hunched posture, slightly ruffled fur and visible slightly to moderate weight loss were noted in one 700 mg/kg bw/day females on two days. Slight to moderate hair loss was noted in one 700 mg/kg bw/day female on the ventral thorax, the abdomen, the genital region as well as the left and right thigh. Slight breathing noises were also noted in one female during some days of this period. On one day of the mating period, slight salivation was noted in one female of this dose group.

No clinical signs were noted during weekly observations in males of all groups during the pre-mating and mating period. In one 700 mg/kg bw/day female, slightly decreased activity and hunched posture were noted during the pre-mating period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In the males, no statistically significant differences of the mean bodyweights and the mean bodyweight gain of males treated with 50 mg/kg of the test material were noted when compared with the control males during the pre-mating and mating period of the study. The bodyweight gain of males treated with 200 mg/kg was decreased (p < 0.01) on day two. The mean bodyweight of males treated with 700 mg/kg was slightly decreased (p < 0.05 or p < 0.01) during days three to give and the mean bodyweight gain was slightly decreased (p<0.01) compared with controls from day two until day 14 of the pre-mating period. This was not noted during the mating period.

In the females, no test material related effects on the mean bodyweights of the females were observed when compared with the controls during the pre-mating, mating, gestation and lactation period.

The mean bodyweight gain of females treated with 700 mg/kg of the test material was slightly decreased compared to the controls during days three to seven (p < 0.05 or p < 0.01) and on days 12 to 14 (p < 0.05 pr p < 0.01) of the pre-mating period. In females treated with 200 mg/kg, a slight decrease (p < 0.05 or p < 0.01) when compared to controls from days 11 to 21 of the gestation period was observed.

The mean food consumption of 700 mg/kg bw/day males was slightly decreased (p<0.05) when compared to controls during week one and increased (p<0.01) during week two. The mean food consumption of males of the other treated groups showed no statistically significant changes. 700 mg/kg bw/day females showed a tendency of a slightly decreased mean absolute and relative food consumption during the pre-mating period. During the first week of the pre-mating period, this decrease of the mean absolute food consumption was statistically significant (p<0.01).

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Degeneration of spermatocytes/spermatids and increased incidence and/or severity of Sertoli cell vacuolation were recorded in testes of group 4 (700 mg/kg). Increase in cellular debris in the tubules, decrease in elongate spermatids and/or loss of germ cells were frequently observed in the affected testes of males in group 4. No abnormalities were identified on the Spermatogonia. Continual stressful condition of the animals suggested by thymic atrophy and/or adrenocortical hypertrophy was expected mainly in animals of group 4 and might have contributed to the testicular lesions, although the possible effects on the spermocytes/spermatids could not be excluded. Degenerative testicular changes recorded in the 700 mg/kg/day, irrespective of its cause were considered to be responsible for female infertility.

Increased cellular debris in the duct lumen as well as oligospermia in epididymides was recorded in males of group 4 (700 mg/kg). These were considered to be secondary effects of the testicular lesions observed in this group. Reduced secretion was recorded in the prostrate glands, seminal vesicle and coagulating glands in males of group 4 as well. There were no indicators of cellular/tissue injuries in these organs, and therefore, these findings were considered to be secondary changes following the testicular lesions, or the poor condition of these animals.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating Performance and Fertility:
Eleven females mated in groups 1, 2 and 3 and 7 or group four were mated within the first or second mating period.

The median and mean pre-coital times were unaffected by treatment with the test material. Mean pre-coital times were 2.9, 2.3, 3.9* and 3.4 days in groups 1, 2, 3 and 4, respectively. The median pre-coital times were 3, 3, 3, and 4 days in order of ascending dose level. For the one female (700 mg/kg /day) that mated during the pre-coital time was 6 days, it is not determined whether the mating was initially overlooked.

The fertility index was 90.9, 81.8, 81.8 and 62.5 % in the 0, 50, 200 and 700 mg/kg/day groups respectively. In the 700 mg/kg/day, the conception rate was similar to the fertility index (71.4 %).

All pregnant females treated with 700 mg/kg/day did not deliver any offspring. The gestation index was 100, 100, 77.8 and 0 % in the 0, 50, 200 and 700 mg/kg/day groups respectively.

Corpora Lutea Count:
The mean number of corpora lutea per dam was similar in all groups, 14.8, 14.2 and 14.1 in the 0, 50 and 200 mg/kg/day groups, the mean number of corpora lutea in the high dose group could not be determined due to the dams not delivering a litter.

Duration of Gestation:
The mean duration of gestation was unaffected by exposure to the test material. Mean duration of gestation was 21.6, 21.4, 21.9 and not determined (no litter delivered) in order of ascending dose level.

Implantation Rate and Post-Implantation Loss:
The mean number of implantations was slightly decreased in females treated with 50 and 200 mg/kg/day, this did not reach statistical significance. The post-implantation loss was increased slightly at the 50 mg/kg/day group, and increased at the 200 mg/kg/day group which reached statistical significance (p < 0.01). This was considered to be test material related.

The mean number of implantation per litter were 14.2, 13.2 and 11.7 at 0, 50 and 200 mg/kg/day. These were all within the historical control range. The number of implantations in the high dose group could not be determined as no litter was delivered.

Litter Size at First Litter Check:
In the 0, 50 and 200 mg/kg/day group, the mean birth indices were slightly decreased in a dose-response pattern (91.5, 84.0 and 61.0 (p < 0.01)). As none of the high dose group females delivered, this could not be calculated for this dose level. This effect was considered to be related to test material administration.

Postnatal Loss Days 0 - 4 Post Partum:
No effects on the postnatal loss between day 0 and 4 post partum were noted in females treated with 50 mg/kg/day or 200 mg/kg/day of the test item. The mean total postnatal loss was 1, 2, 1 and not detectable in groups 1, 2, 3 and 4 respectively.
The viability index was 99.2, 98.0% in groups 1 to 3 and it was not detectable in group 4 due to no delivery.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No statistically significant changes in the mean organ weights, organ to body weight ratios and organ to brain ratios were noted in females when compared with the control animals.

In males, the mean liver weight, the mean liver to body weight and the mean liver to brain weight ratio were increased (p < 0.01) in animals treated with 700 mg/kg/day of the test item when compared with controls. The mean thymus weight, the mean thymus to body weight ratio and the mean thymus to brain weight ratio were decreased (p<0.01) in males of this dose group when compared with controls. Both findings were considered to be test item related.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no gross lesions in the animals that died as a results of treatment with the test material, and the gross changes were considered to be incidental lesions, non-specific changes that are commonly observed in the decedent the lesions are considered to be related to stressful conditions and also spontaneous lesions which are associated with animals of the strain and age used in the study.

Incomplete collapse of the lung was observed in two animals (11 a control male and 31 a male from the 300 mg/kg group). This correlated with observed alveolar oedema (minimal in animal 11 and moderate in animal 31).

In animal number 31, slight mucosal necrosis was observed microscopically in the trachea. These microscopic changes were considered to be due to incidental influx (e.g. regurgitation) of the dosing solution into the respiratory tract, this was considered to be related to the morbidity of this animal.
In animal number 11, incidental influx of the dosing solution into the respiratory tract was suspected to be the cause of the animals morbidity, although histological changes could not be identified on the trachea die to progressive of autolysis.

Discolouration recorded in lungs, thymus and lymph node, which were correlated microscopically with congestion and/or agonal haemorrhage were considered to be non-specific changes commonly observed in the decedent.

The black-foci of the gastric fundus and the reduced splenic size, which were correlated microscopically with glandular stomach erosion and splicing follicular atrophy/reduced red pulp area respectively, were considered to be non-specific, secondary changes associated with a stressful condition.
Liquid and/or brown contents in the stomach and/or intestines were recorded in animals 40, 42 and 78 (2 males from the 700 mg/kg group and one female from the 700 mg/kg group). Such microscopic findings may be observed when there were haemorrhagic lesions in the alimentary tracts. In animal 78 (the female from the 700 mg/kg group), black-contents in the ileum and cecum were recorded. Although there were no histological abnormalities in these organs, glandular stomach erosion with mucosal regeneration was observed microscopically, and therefore, macroscopic findings in the ileum and cecum were considered to be associated with gastric injuries.

In the other two males from this group, microscopic changes that were considered to be attributable to macroscopic findings could not be identified in these animals due to the progression of autolysis.

Splenic constriction and dark red discolouration of the ovary were correlated with focal fibrosis and congestion respectively when examined microscopically. These were considered to be spontaneous changes which may be observed in rats of this strain and age.

Gross lesions in the survivors attributable to treatment with the test item were recorded in the liver and the thymus.

Enlarged liver was recorded in two males of group 1, one male of group 2 and five males of group 4, the incidence was higher in group 4 males.
Reduced thymic size was observed in four males of group 4.

All other gross lesions recorded in the animals surviving to termination were within the normal background alterations for this age and strain of rat.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The test material induced histomorphological changes in testes, epididymides, prostate glands, seminal vesicles, coagulating glands, kidneys, stomach, liver, adrenal glands, thymus and trachea.

In the females, no specific effects were observed in the ovaries.

In the kidney, increased incidence and/or severity of tubular degeneration/regeneration were recorded in both sexes of group 4 (700 mg/kg), these degenerative changes were considered to be adverse.

In the stomach, hyperkeratosis and/or squamous cell hypertrophy (increased mucosal thickness) of the forestomach were recorded in animals of groups 3 and 4 (200 and 700 mg/kg, respectively). In addition to these findings, squamous cell hyperplasia and ulcer were recorded in the forestomach of the animals that died in group 4. These findings were considered to be attributable to the irritating properties of the test material. Hyperkeratosis and squamous cell hypertrophy of the forestomach were considered to be a simple reactive change to the irritative stimuli of the test material and was therefore not considered to be an adverse effect. The observed ulceration and squamous cell hyperplasia were tissue injury and its reparative process and was therefore considered to be adverse.

In the liver, centrilobular hepatocellular hypertrophy was recorded in both sexes of groups 3 and 4 (200 and 700 mg/kg, respectively). This was considered to be related to metabolism and adaptive in character, and there were no further indications of liver injuries. This was therefore not considered to be adverse.

Epithelial hypertrophy and regenerated mucosal epithelium of trachea were sporadically recorded in the survivors of groups 2 and 4. Mucosal necrosis and regenerated mucosal epithelium were also recorded in the decedent of groups 3 and 4. In addition, a slight increase in the group mean severity of mucosal/submucosal inflammatory cell infiltration was recorded in group 4. These were not caused by the systemic toxicity of the test material and were considered to be related to incidental influx (e.g. regurgitation) of dosing solutions containing the test material into the respiratory tract. The irritating properties of the test material was attributed to the slightly higher incidence in severity of the mucosal injuries in the groups treated with the test material compared to the control group.

OTHER FINDINGS (PARENTAL ANIMALS)
Neurobehavioral examination: None of the parameters investigated during the functional observational battery gave an indication of a test material-related effect.

Haematology: No test material related changes in the investigated haematology parameters were noted in males and females when compared with the control animals.

Clinical chemistry: Mean bilirubin was decreased in males treated with 200 and700 mg/kg/day which demonstrated a dose response relationship. Mean triglyceride value of males treated with 700 mg/kg/day was increased. The mean content of albumin was increased, the mean content of globulin was decreased and (mean albumin to globulin ratio was therefore increased) in males treated with 700 mg/kg/day.

The mean values of bilirubin were decreased in females treated with 200 and 700 mg/kg/day.

Mean triglyceride value of females treated with 700 mg/kg/day.

The mean value of protein was decreased in females treated with 200 and 700 mg/kg/day. The mean content of globulin was decreased in females treated with the test material the resulting mean albumin to globulin ratio was increased in females treated with 700 mg/kg/day.
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive effects and parental toxicity
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects at this dose
Critical effects observed:
no
Clinical signs:
not examined
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Litter Data - F1 pups:
External Examination at First Litter Check and during Lactation:
No test material related findings were noted in the first litter check or during the first four days post-partum.

Sex Ratios:
The mean number of pups at the first litter check was decreased in a dose-response pattern. No pups were delivered by females in the high dose group. The ratio of males to females was 50/50 in the 0 mg/kg/day group and was 45/55 and 36/64 in the 50 and 200 mg/kg/day group. A test material effect could not be discounted.

Body Weights to Day 4 Post Partum:
Mean pup weights on day 1 and 4 post-partum were unaffected by maternal treatment. The mean bodyweights of pups in the 50 mg/kg/day group was slightly increased (p < 0.05), however, as it was only seen at this dose level, it was considered incidental.

No test material related macroscopic findings were observed in the pups at necropsy.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see discussions
Critical effects observed:
no
Reproductive effects observed:
not specified

Table 3: Incidence and Severity (mean) of main findings in testes and epididymides

Incidence/Mean Severity

Group 1

Group 2

Group 3

Group 4

Testes

11 M

7 M

7 M

11 M

Degeneration, spermatocytes/spermatids

0

0

0

10/1.5

Sertoli cell vacuolation

2/1.0

1/1.0

0

9/1.8

Epididymides

11 M

7 M

7 M

11 M

Increased cellular debris in the lumen, with/without decreased normal maturing spermatazoa

0

0

1/1.0

9/1.2

Oligospermia

0

0

0

2/4.0

 

Table 4: Sperm staging

Group

Animal No.

Stage

Cell Population

Findings

1

1

Complete

Complete

Sertoli cell vacuolation, unilateral, minimal

2

Complete

Complete

None

3

Complete

Complete

None

4

Complete

Complete

None

5

Complete

Complete

None

6

Complete

Complete

None

7

Complete

Complete

Sertoli cell vacuolation, unilateral, minimal

8

Complete

Complete

None

9

Complete

Complete

Vasculitis, unilateral, minimal

10

Complete

Complete

None

11

Complete#

Complete#

None

2

12

Complete

Complete

Tubular atrophy, focal, unilateral, minimal;

Sertoli cell vacuolation, unilateral, minimal

13

Complete

Complete

None

14

Complete

Complete

None

15

Complete

Complete

None

16

Complete

Complete

None

17

Complete

Complete

None

21

Complete

Complete

None

3

23

Complete

Complete

Vasculitis, unilateral, minimal

24

Complete

Complete

None

25

Complete

Complete

None

26

Complete

Complete

None

27

Complete

Complete

None

28

Complete

Complete

Tubular atrophy, focal, unilateral, slight

31

Complete

Complete

None

4

34

Complete

Complete

Degeneration, spermatocytes/spermatids, bilateral, slight (with increase in cellular debris and decrease in elongated spermatids in tubules, and loss of germ cells);

Sertoli cell vacuolation, bilateral, moderate

35

Complete

Complete

Degeneration, spermatocytes/spermatids, bilateral, slight (with increase in cellular debris and decrease in elongated spermatids in tubules);

Sertoli cell vacuolation, bilateral, slight

36

Complete

Complete

Degeneration, spermatocytes/spermatids, bilateral, slight (with increase in cellular debris and decrease in elongated spermatids in tubules, and loss of germ cells);

Sertoli cell vacuolation, bilateral, slight

37

Complete

Complete

Degeneration, spermatocytes/spermatids, bilateral minimal (with increase in cellular debris in tubules);

Sertoli cell vacuolation, unilateral, minimal

38

Complete

Complete

Degeneration, spermatocytes/spermatids, bilateral, slight (with increase in cellular debris and decrease in elongated spermatids in tubules, and loss of germ cells);

Sertoli cell vacuolation, bilateral, slight

39

Complete

Complete

Degeneration, spermatocytes/spermatids, bilateral, minimal (with increase in cellular debris in tubules);

Sertoli cell vacuolation, bilateral, minimal

40

Complete#

Complete#

Degeneration, spermatocytes/spermatids, bilateral minimal

41

Complete

Complete

Degeneration, spermatocytes/spermatids, bilateral, minimal (with increase in cellular debris in tubules);

Sertoli cell vacuolation, bilateral, slight

42

Complete#

Complete#

None

43

Complete

Complete

Degeneration, spermatocytes/spermatids, bilateral, minimal (with increase in cellular debris in tubules);

Sertoli cell vacuolation, bilateral, minimal

44

Complete

Complete

Degeneration, spermatocytes/spermatids, bilateral, minimal (with increase in cellular debris in tubules);

Sertoli cell vacoulation, bilateral, slight

# Autolysis had begun, but the representative stages/cell populations were identified and the completeness confirmed

 

Table 5: Incidence and Severity (mean) of main findings in prostate, and coagulating glands and seminal vesicles

Incidence/Mean Severity

Group 1 (n = 10)

Group 2 (n = 7)

Group 3 (n = 6_

Group 4 (n = 9)

Prostate gland - Reduced secretion

0

0

0

3/1.0

Seminal vesicles - Reduced secretion

0

0

0

2/1.0

Coagulating glands - Reduced secretion

0

0

0

2/1.0

 

Table 6: Summary of Reproductive Performance

Dose (mg/kg/day)

0

50

200

700

Number of females paired

11

11

11

8

Number of females mated

11

11

11

7

Number of unscheduled deaths

0

0

2

5

Number of females not pregnant

0

0

2

3

Number of females pregnant but did not deliver

0

0

2

5

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

10

9

7

0

 

Conclusions:
Under the conditions of the test, the no-observed-adverse effect level (NOAEL) for reproductive effects and parental toxicity was considered to be 200 mg/kg/day for males and females, and the no-observed effect level (NOEL) was established as 50 mg/kg/day in both sexes.
Executive summary:

The test material was administered to male rats for at east 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum. The test material was applied at 0, 50, 200 and 700 mg/kg bw/day (groups 1 to 4, respectively). The following effects were recorded:

One male in group one, one male and two females of group three and two males and five females of group four died or were killed in extremis prematurely. The cause of death of the male in group one and one male and one female in group three was considered to be incidental influx of the dosing solution into the respiratory tract. In the other animals, the stressful condition attributable to treatment of the test material was considered to be the cause of death.

None of the parameters investigated during the functional observational battery indicated a test material-related effect.

The mean food consumption of 50 and 200 mg/kg bw/day males were not statistically significant when compared to the controls. The mean food consumption of 700 mg/kg males was slightly decreased in week one and slightly increased in week two when compared to the controls. 700 mg/kg bw/day females showed a tendency of a slightly decreased mean absolute and relative food consumption on the pre-pairing period. During the first week of this period, the decreased of the mean absolute food consumption was statistically significant. In 200 mg/kg bw/day females, the mean food consumption was slightly decreased during the lactation period.

No statistically significant differences in mean body weights and mean body weight gain of 50 mg/kg bw/day males were noted compared to the controls during the pre-pairing and pairing period. The mean body weight of 700 mg/kg bw/day males was slightly decreased during days three to five and the mean body weight gain was slightly decreased when compared to the controls from day two to day 14 of the pre-pairing period (this was not noted during the pairing period). No effects on mean body weights of females were observed during the pre-pairing, pairing, gestation and lactation periods. The mean body weight gain of 700 mg/kg bw/day females was slightly decreased compared to controls during days three to seven and on days 12 to 14 of the pre-pairing period. The mean body weight gain was slightly decreased in 200 mg/kg bw/day females when compared to controls from day 11 to 21 of the gestation period.

No changes in haematology parameters were noted in males and females when compared to the controls.

The mean values of bilirubin were decreased in 200 and 700 mg/kg bw/day males when compared to the controls; this decrease showed a dose-response relationship and is therefore considered to be test material related. The mean triglyceride value of 700 mg/kg bw/day males was increased when compared to the controls; this value exceeds the mean standard deviation of the historical reference data and is therefore considered to be test material related. The mean albumin content was increased, the mean globulin content was decreased and, due to this, the mean albumin to globulin ratio was increased in 700 mg/kg bw/day males; it cannot be excluded that this is test material related. The mean bilirubin values decreased in 200 and 700 mg/kg bw/day females when compared to the controls; this decrease showed a dose response relationship and is therefore considered to be test material related. The mean triglyceride value in 700 mg/kg bw/day females was increased when compared to the controls; this value exceeds the mean standard deviation of the historical reference data and is considered to be test material related. The mean protein value decreased in 200 and 700 mg/kg bw/day females when compared to the controls. The mean content of globulin decreased in all treated groups compared to the controls; due to this the mean albumin to globulin ratio increased in 700 mg/kg bw/day females when compared to the controls and it cannot be excluded that this is test material related.

No statistically significant differences in mean organ weights, mean organ to body weight ratios and mean organ to brain weight ratios were noted in treated females when compared to controls. In 700 mg/kg bw/day males, the mean liver weight, the mean liver to body weight ratio and the mean liver to brain weight ratio were statistically significantly increased when compared to controls. The mean thymus weight, the mean thymus to body weight ratio and the mean thymus to brain weight ratio of this dose group were statistically significantly decreased when compared to controls.

In the kidney, increased incidence and/or severity of tubular degeneration/regeneration were recorded in both sexes at 700 mg/kg bw/day and such degenerative lesions were considered to be adverse. In the stomach, hyperkatosis and/or squamous cell hypertrophy were recorded in 200 and 700 mg/kg bw/day animals. In addition to these findings, squamous cell hyperplasia and ulcer were recorded in the forestomach of 700 mg/kg bw/day animals. These findings were considered to be related to the irritant property of the test material.

In the liver, centrilobular hepatocellular hypertrophy was recorded in both sexes of 200 and 700 mg/kg bw/day animals. This was considered to be of metabolic nature and adaptive character and there were no further indicators of liver injuries. Epithelia hypertrophy and regenerated mucosal epithelium of trachea were sporadically recorded in 50 and 700 mg/kg bw/day survivors. Mucosal necrosis and regenerated mucosal epithelium were also recorded in the decedent of 200 and 700 mg/kg bw/day animals. In addition, a slight increase in the group mean severity of mucosal/submucosal inflammatory cell infiltration was recorded in 700 mg/kg bw/day animals. These were considered to be associated with incidental influx of the dosing solution containing the test material.

Degeneration of spermatocytes/spermatids and increased incidence and/or severity of Seroli cell vacuolation were recorded in testes of 700 mg/kg bw/day males. Increase in cellular debris in the tubules, decrease in elongate spermatids and/or loss of germ cells were frequently observed in the affected males in this group. No abnormalities were identified in spermatogonia. Continual stressful condition being suggested by thymic atrophy and/or adrenocortical hypertrophy was expected mainly at 700 mg/kg bw/day and might have contributed to the testicular lesions although the possible direct effects of the test material on spermatocytes/spermatids could not be excluded. Degenerative testicular changes recorded in group four animals, irrespective of the cause, were considered to be responsible for female infertility and considered to be adverse. Increased cellular debris in the duct lumen as well as oligospermia in epididymides was recorded in 700 mg/kg bw/day males. These were considered to have been secondary events following testicular lesions. Reduced secretion was recorded in the prostate glands, seminal vesicles and coagulating glands in 700 mg/kg bw/day males. There were no indicators of cellular/tissue injuries in these organs and, therefore, these findings were considered to be secondary changes following the testicular lesions caused by worsened general condition.

The mean precoital time was not affected by the treatment with the test material at any dose level. The fertility index was slightly decreased in animals treated with 700 mg/kg/day in comparison to controls. The post-implantation loss was slightly increased in females treated with 50 mg/kg/day and in females treated with 200 mg/kg/day of the test material when compared with controls. In the 0, 50 and 200 mg/kg/day groups, the mean birth indices were slightly decreased in a dose response pattern. The indices were 91.5, 84.0, 61.0 and the index was not detectable in group 4 due to no delivery. The mean litter size at first litter check was also decreased with increasing doses 13.0, 11.1, 7.1 and not detectable in group 4 due to no delivery. These findings were considered to be test material related.

 

The mean number of pups at first litter observation was decreased in a dose-response. No pups were delivered by females treated with 700 mg/kg/day. The ratio of males to females was 50/50 in the control group this appeared to be skewed by test material administration to to 45/55 in the group treated with 50 mg/kg/day to 36/64 in the group treated with 200 mg/kg/day. It could be not excluded that this shift was test material related. No test material-related differences in the mean body weight development of pups were noted in the 50 mg/kg/day or 200 mg/kg/day groups when compared to the controls. At necropsy, there were no abnormal findings in the pups.

 

Under the conditions of the test, the no-observed-adverse effect level (NOAEL) for reproductive effects and parental toxicity was considered to be 200 mg/kg/day for males and females, and the no-observed effect level (NOEL) was established as 50 mg/kg/day in both sexes.

Data source

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

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
(Z)-3-hexenyl salicylate
EC Number:
265-745-8
EC Name:
(Z)-3-hexenyl salicylate
Cas Number:
65405-77-8
Molecular formula:
C13H16O3
IUPAC Name:
(3Z)-hex-3-en-1-yl 2-hydroxybenzoate
Test material form:
liquid
Details on test material:
- Physical state: liquid
- Stability under test conditions: yes
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst, The Netherlands
- Weight at study initiation: 291 - 367 g (males); 189 - 239 g (females)
- Housing: individually in Makrolon type-3 cages with wire mesh tops
- Diet: Pelleted standard Harlan Teklad 2914C ad libitum
- Water: tap water ad libitum
- Acclimatisation 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: 6th September 2012: To: 15th October 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was weighed into a glass beaker and the vehicle added. The mixtures were stirred using a magnetic stirrer and stored at room temperature.

VEHICLE
- Lot/batch no. (if required): BCBG8285V
- Dose Volume: 5 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulations were analysed by GC-FID.

Concentration, homogeneity and stability were determined in samples taken after experimental start (see Table 1 in 'Any other information on materials and methods incl. tables').

Concentration and homogeneity were further determined (see Table 2 in 'Any other information on materials and methods incl. tables').
Duration of treatment / exposure:
males: 4 weeks; females: 7 weeks
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group (I)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
700 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
11
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: previous non-GLP range-finding study (details below):
14-day dose-range finding oral toxicity (gavage) study was performed to select doses for OECD 422 study.
Discussion and conclusion on 14-day study:
Oral administration to Wistar rats at doses of 100, 300 and 1000 mg/kg, for 14 days resulted in:
Mortality of three males treated with 1000 mg/kg/day, which were found spontaneously dead on treatment days five or six, four females of this dose group, which were found spontaneously dead on treatment days give or seven and one female of this group, which was sacrificed in extremis on day seven.
In males and females treated with 100 mg/kg/day or 300 mg/kg/day of the test item, no mortality, no test item-related clinical signs, no changes in mean absolute and relative food consumption and no differences of toxicological relevance in mean body weights as well as mean body weight gain and no test item related macroscopic findings were noted.
In femlaes treated with 300 mg/kg/day, reduced thymus weight, thymus to body weight ratio and thymus to brain weight ratio was noted with different statistical significances. The mean liver weight, the mean liver to bodyweight ratio and the mean liver to brain weight ratio were also increased in females of this dose group. In females treated with 100 mg/kg/day the mean liver weight, the mean liver to body weight rato and mean liver to brain weight ratio were slightly increased.

Based on the results, doses of 100 and 300 mg/kg/day of the test item could be considered appropriate for subsequent 28-day repeat-dose toxicity and/or reproduction/developmental screening tests in rats. the dose of 1000 mg/kg/day could nt be proposed for a longer term study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
Observations: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity. Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes or bizarre behaviour were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: weekly (males); pre-pairing period days 1 - 8 and 8 - 13 (females); gestation days 0 - 7, 7 - 14 and 14 - 21
post coitum, and days 1 - 4 post partum.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the pre-pairing period.
- Anaesthetic used for blood collection: Yes (light isoflurane)
- Animals fasted: Yes
- How many animals: five males and five females from each group
- Parameters checked: erythrocyte count, haemoglobin, hematocrit, mean corpuscular volume, red cell distribution width, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, total leukocyte count, differential leukocyte count, platelet count, prothrombin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the pre-pairing period.
- Anaesthetic used for blood collection: Yes (light isoflurane)
- Animals fasted: Yes
- How many animals: five males and five females from each group
- Parameters checked: glucose, urea, creatinine, total bilirubin, total cholesterol, triglycerides, aspartate aminotransferase, alanine aminotransferase, akaline phosphatase, gamma-glutamyl-transferase, bile acids, sodium, potassium, chloride, calcium, phosphorus, total protein, albumin, globulin and albumin/globulin ratio.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
At one time during the study (males shortly before sacrifice and females on day 3 or 4 post partum), relevant parameters in a modified Irwin screen test were performed with five parental males and five parental females. Additionally, locomotor activity was measured quantitatively for the same animals using an Activity Monitor. Activity of the animals (based on beam count) was recorded for 10 minute intervals over a 60 minute period.
Sacrifice and pathology:
All animals sacrificed or found dead were weighed and subjected to a detailed macroscopic examination to establish the cause of death. All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

GROSS PATHOLOGY: Yes
- At sacrifice, the testes and epididymides of all parental males were weighed separately. In addition, the following organs were selected from five males and five females from each group and weighed: adrenals, brain, heart, kidneys, liver, thymus and spleen.

HISTOPATHOLOGY: Yes
- The following tissues were examined: prostrate, seminal vesicles with coagulating gland, testes and epididymides (males); ovaries (females); gross lesions, brain, spinal cord, heart, thymus, thyroids and parathyroids, small and large intestines, stomach, liver, kidneys, adrenals, spleen, trachea and lungs, uterus, urinary bladder, lymph nodes, peripheral nerve and bone marrow (males and females).

- Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure. If test material-related morphologic changes were detected in organs of high-dose animals, the same organs from mid-dose and low-dose animals were examined to establish a no-effect level if possible. Histological examination of ovaries was carried out on any females that did not give birth. Microscopic examination of the reproductive organs of all infertile males was made.
Statistics:
The following statistical methods were used to analyse food consumption, body weights and reproduction data:
- Mean and standard deviations were calculated.
- The Dunnett-test based on 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 was applied instead of the Dunnett test when the could not be assumed to follow a normal distribution.
- 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, non-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:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
One control male was found spontaneously dead on day five and one 200 mg/kg bw/day male was found spontaneously dead on day 11; both deaths were considered to be due to an incidental influx of dosing solution into the respiratory tract. Two 700 mg/kg bw/day males were found spontaneously dead on days five and six of the pre-pairing period.

Two 200 mg/kg bw/day females were found spontaneously dead on days 23 and 24 of the gestation period; the death of one of them was considered to be due to incidental influx of dosing solution into the respiratory tract. Three 700 mg/kg bw/day females were found spontaneously dead on days five, six and seven of the pre-pairing period, one female of this dose group was sacrificed in extremis on day two of the pre-pairing period and another female of this group was sacrificed in extremis on day 18 of the gestation period.

Apart from the deaths considered to be due to incidental influx of dosing solution into the respiratory tract, the death of one 200 mg/kg bw/day female was considered to probably be pregnancy toxemia but this was not considered to be directly attributable to the test material. In the remainder of the animals, the worsened general condition which was caused by the effects on the forestomach and kidneys and the continual stressful condition attributable to treatment with the test material were considered to be the cause of death.

No clinical signs were noted in control and 50 mg/kg bw/day males during the pre-pairing period. Slight breathing noises were noted in a few 200 and 700 mg/kg bw/day males on one or two days of the pre-pairing period. During the pairing period, slightly soft faeces were noted in a few control males on one day. Slight breathing noises were noted in a few 50 mg/kg bw/day males on two days and in some 200 and 700 mg/kg bw/day males on some days of the pairing period.

No clinical signs were noted in control, 50 and 200 mg/kg bw/day females during the pre-pairing period. Slight breathing noises were noted in 700 mg/kg bw/day females on some days of the pre-pairing period. Prostration and hunched posture were noted in some 700 mg/kg bw/day females on single days. Additionally, slightly ruffled fur and visible slight weight loss were seen in 700 mg/kg bw/day females on some days, slight convulsion was noted in one female on on day and slightly decreased activity was noted in a few females on one day of the pre-pairing period.

During the pairing period, no clinical signs were noted in control, 50 and 200 mg/kg bw/day females. Slightly decreased activity, hunched posture, slightly ruffled fur and visible slightly to moderate weight loss were noted in one 700 mg/kg bw/day females on two days. Slight to moderate hair loss was noted in one 700 mg/kg bw/day female on the ventral thorax, the abdomen, the genital region as well as the left and right thigh. Slight breathing noises were also noted in one female during some days of this period. On one day of the pairing period, slight salivation was noted in one female of this dose group.

No clinical signs were noted during weekly observations in males of all groups during the pre-pairing and pairing period. In one 700 mg/kg bw/day female, slightly decreased activity and hunched posture were noted during the pre-pairing period.

BODY WEIGHT AND WEIGHT GAIN
In the males, no statistically significant differences of the mean bodyweights and the mean bodyweight gain of males treated with 50 mg/kg of the test material were noted when compared with the control males during the pre-mating and mating period of the study. The bodyweight gain of males treated with 200 mg/kg was decreased (p < 0.01) on day two. The mean bodyweight of males treated with 700 mg/kg was slightly decreased (p < 0.05 or p < 0.01) during days three to give and the mean bodyweight gain was slightly decreased (p<0.01) com[pared with controls from day two until day 14 of the pre-mating period. This was not noted during the mating period.
In the females, no test material related effects on the mean bodyweights of the females were observed when compared with the controls during the pre-mating, mating, gestation and lactation period.
The mean bodyweight gain of females treated with 700 mg/kg of the test material was slightly decreased compared to the controls during days three to sever (p < 0.05 or p < 0.01) and on days 12 to 14 (p < 0.05 pr p < 0.01) of the pre-mating period. In females treated with 200 mg/kg, a slight decrease (p < 0.05 or p < 0.01) when compared to controls from days 11 to 21 of the gestation period was observed.

FOOD CONSUMPTION
The mean food consumption of 700 mg/kg bw/day males was slightly decreased (p<0.05) when compared to controls during week one and increased (p<0.01) during week two. The mean food consumption of males of the other treated groups showed no statistically significant changes. 700 mg/kg bw/day females showed a tendency of a slightly decreased mean absolute and relative food consumption during the pre-pairing period. During the first week of the pre-pairing period, this decrease of the mean absolute food consumption was statistically significant (p<0.01).

HAEMATOLOGY
Slightly decreased (p < 0.05) mean haemoglobin values were noted in males treated with 700 mg/kg when compared with the controls after 14 days of treatment. Additionally, the mean red cell volume distribution width was slightly increased (p < 0.01) in males of this dose group when compared with the controls. These values were within the historical control range and were therefore considered not to be related to the test material.
The mean count of total leucocytes was slightly decreased with (p < 0.05) or without statistical significance in males treated with the test material. This was without any dose response relationship and was therefore not considered to be related to administration of the test material.
The mean absolute and/or relative basophils were slightly decreased with different (p < 0.05 or p < 0.01) or without statistical significance in all groups treated with the test material. This was also without a dose-response relationship and therefore not considered to be test material related. The mean values of absolute monocytes and large unstained cells were slightly decreased (p < 0.05) in males treated with the test material The mean value of absolute lymphocytes was slightly decreased (p < 0.05) in males treated with 700 mg/kg of the test material when compared to controls. All these values were found to be within the historical control range and was therefore not attributed to the administration of the test material.
In females, slightly decreased haemoglobin values (p < 0.01), slightly decreased mean content of erythrocytes (p < 0.05) and a slightly decreased haematocrit value (p < 0.01) were noted in females treated at 700 mg/kg when compared to the controls after 14 days of treatment. Additionally, the mean red cell volume distribution width was slightly increased (p < 0.01) in females of this dose group. The mean values of absolute and relative basophils were slightly decreased (p < 0.05) in females treated with 700 mg/kg, the mean values of absolute and relative eosinophils were slightly decreased (p < 0.05) in females treated with 200 mg/kg, and the mean values of absolute large unstained cells were slightly decreased (p < 0.05 or p < 0.01) in females treated with the test material when compared to the controls. The mean value of absolute netrophils was increased (p < 0.01) in the 700 mg/kg group when compared with controls. All these values were within the range of the historical control and therefore could not be considered to be test related.

CLINICAL CHEMISTRY
The mean values of bilirubin were decreased (p < 0.01) in males treated with 200 mg/kg or 700 mg/kg of the test material when compared to controls. This did not have a dose-response pattern and was therefore not considered to be test material related.
The mean triglyceride value of males treated with 700 mg/kg was increased (p < 0.01) when compared with controls. This value exceeded the mean standard deviation of the historical control and was therefore considered an effect of the test material.
The mean content of albumin was increased (p < 0.01), the mean content of globulin was decreased (p < 0.01) and due to this the mean albumin to globulin ratio was increased (p < 0.05) in males treated with 700 mg/kg. It could not be excluded that this was a test material effect.
The mean content of potassium was slightly decreased (p < 0.05) in males treated with 700 mg/kg. Slightly decreased (p < 0.01 or p < 0.05) values of the mean cholesterol in males treated with 200 mg/kg or 700 mg/kg were noted when compared with controls. The mean content of calcium was slightly decreased (p < 0.01) in males treated with 700 mg/kg. The values of these three parameters (potassium, cholesterol and calcium) were within the historical control range and therefore were not attributed to effects of the test material.
In females, the mean values of bilirubin were decreased (p< 0.01) at 200 mg/kg or 700 mg/kg. This decrease showed a dose-response relationship and was therefore attributed to the test material
The mean triglyceride value for females treated with 700 mg/kg was increased (p < 0.01) when compared with controls. This value exceeded the mean standard deviation of the historical control range and was also considered to be an effect of the test material.
The mean value of protein was decreased in females at 200 mg/kg or 700 mg/kg (p < 0.05 and p < 0.01) when compared with controls. The mean content of globulin was decreased (p < 0.05 or p < 0.01) in all groups treated with the test material. This increase was marked in females at 700 mg/kg which resulted in an increase in the mean albumin to globulin ratio (p < 0.05) when compared with controls. This could not be excluded as a test material effect.
The mean cholesterol value was slightly decreased (p < 0.05) in 700 mg/kg females. The mean content of calcium was slightly decreased (p < 0.01) and the mean phosphorous content was increased (p < 0.05) in this group also. These values were within the historical control range and were therefore not attributed to effects of the test material.

NEUROBEHAVIOUR
None of the parameters investigated during the functional observational battery gave an indication of a test material-related effect. Mean values of fore hand grip strength of 200 and 700 mg/kg bw/day males were slightly decreased when compared with controls. No differences in mean hind limb grip strength of treated males were noted when compared to controls. No differences in mean fore- and hind limb grip strength of treated females were noted when compared to controls.

No statistically significant effects on locomotor activity of treated males and females were noted.

ORGAN WEIGHTS
No statistically significant changes in the mean organ weights, organ to body weight ratios and organ to brain ratios were noted in females when compared with the control animals.
In males, the mean liver weight, the mean liver to body weight and the mean liver to brain weight ratio were increased (p < 0.01) in animals treated with 700 mg/kg/day of the test item when compared with controls. The mean thymus weight, the mean thymus to body weight ratio and the mean thymus to brain weight ratio were decreased (p<0.01) in males of this dose group when compared with controls. Both findings were considered to be test item related.

GROSS PATHOLOGY
There were no gross lesions in the animals that died as a results of treatment with the test material, and the gross changes were considered to be incidental lesions, non-specific changes that are commonly observed in the decedent the lesions are considered to be related to stressful conditions and also spontaneous lesions which are associated with animals of the strain and age used in the study.
Incomplete collapse of the lung was observed in two animals (11 a control male and 31 a male from the 200 mg/kg group). This correlated with observed alveolar oedema (minimal in animal 11 and moderate in animal 31).
In animal number 31, slight mucosal necrosis was observed microscopically in the trachea. These microscopic changes were considered to be due to incidental influx (e.g. regurgitation) of the dosing solution into the respiratory tract, this was considered to be related to the morbidity of this animal.
In animal number 11, incidental influx of the dosing solution into the respiratory tract was suspected to be the cause of the animals morbidity, although histological changes could not be identified on the trachea die to progressive of autolysis.
Discolouration recorded in lungs, thymus and lymph node, which were correlated microscopically with congestion and/or agonal haemorrhage were considered to be non-specific changes commonly observed in the decedent.
The black-foci of the gastric fundus and the reduced splenic size, which were correlated microscopically with glandular stomach erosion and splicing follicular atrophy/reduced red pulp area respectively, were considered to be non-specific, secondary changes associated with a stressful condition.
Liquid and/or brown contents in the stomach and/or intestines were recorded in animals 40, 42 and 78 (2 males from the 700 mg/kg group and one female from the 700 mg/kg group). Such microscopic findings may be observed when there were haemorrhagic lesions in the alimentary tracts. In animal 78 (the female from the 700 mg/kg group), black-contents in the ileum and cecum were recorded. Although there were no histological abnormalities in these organs, glandular stomach erosion with mucosal regeneration was observed microscopically, and therefore, macroscopic findings in the ileum and cecum were considered to be associated with gastric injuries.
In the other two males from this group, microscopic changes that were considered to be attributable to macroscopic findings could not be identified in these animals due to the progression of autolysis.
Splenic constriction and dark red discolouration of the ovary were correlated with focal fibrosis and congestion respectively when examined microscopically. These were considered to be spontaneous changes which may be observed in rats of this strain and age.
Gross lesions in the survivors attributable to treatment with the test item were recorded in the liver and the thymus.
Enlarged liver was recorded in two males of group 1, one male of group 2 and five males of group 4, the incidence was higher in group 4 males.
Reduced thymic size was observed in four males of group 4.
All other gross lesions recorded in the animals surviving to termination were within the normal background alterations for this age and strain of rat.

HISTOPATHOLOGY: NON-NEOPLASTIC
The test material induced histomorphological changes in testes, epididymides, prostate glands, seminal vesicles, coagulating glands, kidneys, stomach, liver, adrenal glands, thymus and trachea.
Degeneration of spermatocytes/spermatids and increased incidence and/or severity of Sertoli cell vacuolation were recorded in testes of group 4 (700 mg/kg). Increase in cellular debris in the tubules, decrease in elongate spermatids and/or loss of germ cells were frequently observed in the affected testes of males in group 4. No abnormalities were identified on the Spermatogonia. Continual stressful condition of the animals suggested by thymic atrophy and/or adrenocortical hypertrophy was expected mainly in animals of group 4 and might have contributed to the testicular lesions, although the possible effects on the spermocytes/spermatids could not be excluded.
Increased cellular debris in the duct lumen as well as oligospermia in epididymides was recorded in males of group 4 (700 mg/kg). These were considered to be secondary effects of the testicular lesions observed in this group. Reduced secretion was recorded in the prostate glands, seminal vesicle and coagulating glands in males of group 4 as well. There were no indicators of cellular/tissue injuries in these organs, and therefore, these findings were considered to be secondary changes following the testicular lesions, or the poor condition of these animals.
In the females, no specific effects were observed in the ovaries.
In the kidney, increased incidence and/or severity of tubular degeneration/regeneration were recorded in both sexes of group 4 (700 mg/kg), these degenerative changes were considered to be adverse.
In the stomach, hyperkeratosis and/or squamous cell hypertrophy (increased mucosal thickness) of the forestomach were recorded in animals of groups 3 and 4 (200 and 700 mg/kg, respectively). In addition to these findings, squamous cell hyperplasia and ulcer were recorded in the forestomach of the animals that died in group 4. These findings were considered to be attributable to the irritating properties of the test material. Hyperkeratosis and squamous cell hypertrophy of the forestomach were considered to be a simple reactive change to the irritative stimuli of the test material and was therefore not considered to be an adverse effect. The observed ulceration and squamous cell hyperplasia were tissue injury and its reparative process and was therefore considered to be adverse.
In the liver, centrilobular hepatocellular hypertrophy was recorded in both sexes of groups 3 and 4 (200 and 700 mg/kg, respectively). This was considered to be related to metabolism and adaptive in character, and there were no further indications of liver injuries. This was therefore not considered to be adverse.
Epithelial hypertrophy and regenerated mucosal epithelium of trachea were sporadically recorded in the survivors of groups 2 and 4. Mucosal necrosis and regenerated mucosal epithelium were also recorded in the decedent of groups 3 and 4. In addition, a slight increase in the group mean severity of mucosal/submucosal inflammatory cell infiltration was recorded in group 4. These were not caused by the systemic toxicity of the test material and were considered to be related to incidental influx (e.g. regurgitation) of dosing solutions containing the test material into the respiratory tract. The irritating properties of the test material was attributed to the slightly higher incidence in severity of the mucosal injuries in the groups treated with the test material compared to the control group.

HISTORICAL CONTROL DATA (if applicable)
Historical control data was included for comparison of haematolocy and clinical biochemistry parameters.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mortality
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mortality; body weight; clinical chemistry; gross pathology; organ weights; histopathology

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 3: Food consumption in the pre-pairing period (g/animal/day)

     0 mg/kg  50 mg/kg  200 mg/kg  700 mg/kg
 Males          
 Days 1 to 8  Mean  21.8  22.8  23.1  16.8*
   S.D.  4.5  1.9  2.2  7.0
   n  11  11  11  11
 Days 8 to 13  Mean  23.9  23.4  23.6  27.3**
   S.D.  1.7  2.3  2.9  2.3
   n  10  11  11  9
 Mean of means    22.9  23.1  23.3  22.1
 Females          
 Days 1 to 8  Mean  17.4  17.2  16.8  12.0**
   S.D.  2.3  1.5  1.4  5.1
   n  11  11  11  11
 Days 8 to 13  Mean  17.5  17.7  17.2  17.8
   S.D.  2.3  1.5  1.2  5.1
   n  11  11  11  8
 Mean of means    17.5  17.4  17.0  14.9

Dunnett's

Table 4: Mean Bodyweights and Bodyweight Gain of Males

Period

Day

0 mg/kg (control)

50 mg/kg

200 mg/kg

700 mg/kg

BW (g)

BW gain (%)

BW (g)

BW gain (%)

BW (g)

BW gain (%)

BW (g)

BW gain (%)

Pre-mating

1

327

0

321

0

323

0

324

0

2

332

1

323

1

321

0**

319

-2**

3

333

2

326

1

326

1

308*

-5**

4

336

3

328

2

328

2

303**

-6**

5

336

4

331

3

332

3

306*

-5**

6

338

4

334

4

333

3

311

-2**

7

338

4

335

4

335

4

314

-1**

8

344

6

338

5

337

4

318

0**

9

344

6

340

6

339

5

321

1**

10

348

7

343

7

342

6

324

2**

11

349

8

344

7

342

6

332

1**

12

352

9

346

8

346

7

324

2**

13

354

9

349

9

350

8

326

3**

14

346

7

342

6

342

5

319

0**

Mating period

1

350

0

344

0

346

0

323

0

2

352

1

347

1

347

0

326

1

3

351

0

349

1

349

1

329

2

4

351

0

349

1

348

1

328

1

5

358

2

355

3

352

2

334

3

6

359

3

356

3

352

2

338

5

7

362

3

358

4

355

3

341

5

8

364

4

360

5

357

3

342

6

9

367

5

362

5

360

4

343

6

10

365

4

363

6

358

4

345

6

11

367

5

367

7

359

4

343

6

12

372

6

371

8

364

5

348

8

13

374

7

372

8

366

6

352

9

14

374

7

373

8

366

6

351

9

15

376

8

376

9

369

7

351

9

BW = Bodyweight

-/**Dunnet-test based on pooled variance * p < 0.05 ** p < 0.01

 

Table 5: Mean Bodyweights and Bodyweight Gain of Females

Period

Day

0 mg/kg (control)

50 mg/kg

200 mg/kg

700 mg/kg

BW (g)

BW gain (%)

BW (g)

BW gain (%)

BW (g)

BW gain (%)

BW (g)

BW gain (%)

Pre-mating

1

213

0

212

0

212

0

212

0

2

214

1

213

0

210

-1

211

-1

3

217

2

214

1

213

1

204

-4**

4

218

2

219

3

216

2

201*

-5**

5

218

3

217

2

216

2

206

-4**

6

219

3

217

3

215

2

206

-3**

7

220

4

218

3

218

3

213

0*

8

222

5

222

5

219

4

216

1

9

221

4

221

4

220

4

217

2

10

222

4

222

5

220

4

213

0

11

225

6

224

6

223

5

217

2

12

226

7

226

7

222

5

214

1**

13

225

6

226

7

224

6

214

1*

14

222

4

223

5

222

5

204

-4**

Gestation period

0

228

0

227

0

233

0

-

-

1

233

2

232

2

236

1

-

-

2

240

5

238

5

241

4

-

-

3

243

6

239

6

244

5

-

-

4

247

8

245

8

247

6*

-

-

5

251

10

249

10

251

8

-

-

6

254

11

253

12

255

10

-

-

7

257

12

255

13

258

11

-

-

8

262

15

260

15

261

12

-

-

9

264

16

263

16

263

13

-

-

10

268

18

266

17

268

15

-

-

11

276

21

273

20

273

17*

-

-

12

280

23

277

22

276

19*

-

-

13

284

25

281

24

280

20*

-

-

14

287

26

285

26

284

22*

-

-

15

294

29

291

28

289

24*

-

-

16

303

33

301

33

296

27*

-

-

17

316

38

312

38

304

31**

-

-

18

329

44

323

43

315

35**

-

-

19

342

50

336

48

326

40**

-

-

20

355

56

348

54

337

45**

-

-

21

364

59

356

57

344

48**

-

-

Lactation period

1

267

0

270

0

269

0

2

268

1

270

0

270

0

3

273

2

273

1

272

1

4

276

4

275

2

272

1

BW = Bodyweight

-/**Dunnet-test based on pooled variance * p < 0.05 ** p < 0.01

 

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test, the no-observed-adverse effect level (NOAEL) was considered to be 200 mg/kg/day for males and females, and the no-observed effect level (NOEL) was established as 50 mg/kg/day in both sexes.
Executive summary:

The test material was administered to male rats for at least 28 days and to female rats for 14 days prior to mating, through the mating and gestation periods until the F1 generation reached day 4 post partum. The test material was applied at 0, 50, 200 and 700 mg/kg bw/day (groups 1 to 4, respectively). The following effects were noted:

One male in group one, one male and two females of group three and two males and five females of group four died or were killed in extremis prematurely. The cause of death of the male in group one and one male and one female in group three was considered to be incidental influx of the dosing solution into the respiratory tract. In the other animals, the stressful condition attributable to treatment of the test material was considered to be the cause of death.

None of the parameters investigated during the functional observational battery indicated a test material-related effect.

The mean food consumption of 50 and 200 mg/kg bw/day males were not statistically significant when compared to the controls. The mean food consumption of 700 mg/kg males was slightly decreased in week one and slightly increased in week two when compared to the controls. 700 mg/kg bw/day females showed a tendency of a slightly decreased mean absolute and relative food consumption on the pre-pairing period. During the first week of this period, the decreased of the mean absolute food consumption was statistically significant. In 200 mg/kg bw/day females, the mean food consumption was slightly decreased during the lactation period.

No statistically significant differences in mean body weights and mean body weight gain of 50 mg/kg bw/day males were noted compared to the controls during the pre-pairing and pairing period. The mean body weight of 700 mg/kg bw/day males was slightly decreased during days three to five and the mean body weight gain was slightly decreased when compared to the controls from day two to day 14 of the pre-pairing period (this was not noted during the pairing period). No effects on mean body weights of females were observed during the pre-pairing, pairing, gestation and lactation periods. The mean body weight gain of 700 mg/kg bw/day females was slightly decreased compared to controls during days three to seven and on days 12 to 14 of the pre-pairing period. The mean body weight gain was slightly decreased in 200 mg/kg bw/day females when compared to controls from day 11 to 21 of the gestation period.

No changes in haematology parameters were noted in males and females when compared to the controls.

The mean values of bilirubin were decreased in 200 and 700 mg/kg bw/day males when compared to the controls; this decrease showed a dose-response relationship and is therefore considered to be test material related. The mean triglyceride value of 700 mg/kg bw/day males was increased when compared to the controls; this value exceeds the mean standard deviation of the historical reference data and is therefore considered to be test material related. The mean albumin content was increased, the mean globulin content was decreased and, due to this, the mean albumin to globulin ratio was increased in 700 mg/kg bw/day males; it cannot be excluded that this is test material related. The mean bilirubin values decreased in 200 and 700 mg/kg bw/day females when compared to the controls; this decrease showed a dose response relationship and is therefore considered to be test material related. The mean triglyceride value in 700 mg/kg bw/day females was increased when compared to the controls; this value exceeds the mean standard deviation of the historical reference data and is considered to be test material related. The mean protein value decreased in 200 and 700 mg/kg bw/day females when compared to the controls. The mean content of globulin decreased in all treated groups compared to the controls; due to this the mean albumin to globulin ratio increased in 700 mg/kg bw/day females when compared to the controls and it cannot be excluded that this is test material related.

No statistically significant differences in mean organ weights, mean organ to body weight ratios and mean organ to brain weight ratios were noted in treated females when compared to controls. In 700 mg/kg bw/day males, the mean liver weight, the mean liver to body weight ratio and the mean liver to brain weight ratio were statistically significantly increased when compared to controls. The mean thymus weight, the mean thymus to body weight ratio and the mean thymus to brain weight ratio of this dose group were statistically significantly decreased when compared to controls.

In the kidney, increased incidence and/or severity of tubular degeneration/regeneration were recorded in both sexes at 700 mg/kg bw/day and such degenerative lesions were considered to be adverse. In the stomach, hyperkatosis and/or squamous cell hypertrophy were recorded in 200 and 700 mg/kg bw/day animals. In addition to these findings, squamous cell hyperplasia and ulcer were recorded in the forestomach of 700 mg/kg bw/day animals. These findings were considered to be related to the irritant property of the test material.

In the liver, centrilobular hepatocellular hypertrophy was recorded in both sexes of 200 and 700 mg/kg bw/day animals. This was considered to be of metabolic nature and adaptive character and there were no further indicators of liver injuries. Epithelia hypertrophy and regenerated mucosal epithelium of trachea were sporadically recorded in 50 and 700 mg/kg bw/day survivors. Mucosal necrosis and regenerated mucosal epithelium were also recorded in the decedent of 200 and 700 mg/kg bw/day animals. In addition, a slight increase in the group mean severity of mucosal/submucosal inflammatory cell infiltration was recorded in 700 mg/kg bw/day animals. These were considered to be associated with incidental influx of the dosing solution containing the test material.

Degeneration of spermatocytes/spermatids and increased incidence and/or severity of Sertoli cell vacuolation were recorded in testes of 700 mg/kg bw/day males. Increase in cellular debris in the tubules, decrease in elongated spermatids and/or loss of germ cells were frequently observed in the affected males in this group. No abnormalities were identified in spermatogonia. Continual stressful condition being suggested by thymic atrophy and/or adrenocortical hypertrophy was expected mainly at 700 mg/kg bw/day and might have contributed to the testicular lesions although the possible direct effects of the test material on spermatocytes/spermatids could not be excluded. Degenerative testicular changes recorded in group four animals, irrespective of the cause, were considered to be responsible for female infertility and considered to be adverse. Increased cellular debris in the duct lumen as well as oligospermia in epididymides was recorded in 700 mg/kg bw/day males. These were considered to have been secondary events following testicular lesions. Reduced secretion was recorded in the prostate glands, seminal vesicles and coagulating glands in 700 mg/kg bw/day males. There were no indicators of cellular/tissue injuries in these organs and, therefore, these findings were considered to be secondary changes following the testicular lesions caused by worsened general condition.

The NOAEL was considered to be 200 mg/kg bw/day for both sexes and the NOEL was considered to be 50 mg/kg bw/day for both sexes.