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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 3 JAN 2007 to 22 FEB 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 422)
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

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:
yes
Remarks:
but according to the study authors none of these deviations affected the study's integrity (for details please see "Any other information on materials and methods incl tables").
GLP compliance:
yes
Remarks:
according to OECD GLP Guidelines (1997)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Cyclohex-1,2-ylenediamine
EC Number:
211-776-7
EC Name:
Cyclohex-1,2-ylenediamine
Cas Number:
694-83-7
Molecular formula:
C6H14N2
IUPAC Name:
cyclohexane-1,2-diamine
Details on test material:
- Name of test material (as cited in study report): Dytek® DCH-99, 1,2-Diaminocyclohexane (DCH)
- Substance type: clear light-yellow
- Physical state: liquid
- Analytical purity: 99.5%
- Composition of test material, percentage of components:
99.55% 1,2-Diaminocyclohexane (CAS 694-83-7)
0.24% 2-Aminomethylcyclopentylamine
(CAS 21544-02-5)
0.08% Hexamethyleneimine (CAS 111-49-9)
0.00% Hexamethylenediamine (CAS 124-09-4)
0.07% 2-Methy11,5-pentamethylenediamine
(CAS 15520-10-2)
0.06% Water
- Stability under test conditions: test material in vehicle is stable for at least 5 hours when stored at room temperature
- Storage condition of test material: at ambient temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: (P) approx. 10 wks
- Weight at study initiation: (P) Males mean: 336.75 g; Females mean: 229 g
- Housing:
#Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
#Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
#Post-mating: Males were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm). Females were individually housed in Macrolon cages (MIII type, height 18 cm).
#Lactation: Offspring was kept with the dam until termination.
General Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives. During overnight activity monitoring, animals were housed individually in Macrolon cages (MIII type; height 15 cm) with sterilised sawdust as bedding material. No cage-enrichment was provided during overnight activity monitoring.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest,Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2-23.3
- Humidity (%): 29-87%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 8 JAN 2007 To: 22 FEB 2007

FURTHER DETAILS:
Animals were housed in Room 16 until 09 January 2007 and in Room 3 from 09 January 2007 (2nd day of substance treatment) onwards.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Milli-U from Millipore Corporation, Bedford, USA
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the test substance.
- Rationale for vehicle: Based an trial formulations performed at NOTOX.
Details on mating procedure:
- M/F ratio per cage:1/1
- Length of cohabitation: maximum 14 days
- Proof of pregnancy: sperm in vaginal lavage referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing seperation of animals. But on day 15 the same male was placed back with the female until mating was detected.Mating continued until the day of necropsy of the males (Day 18 of mating).
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually housed in Macrolon cages (MIII type, height 18 cm).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Group Analysis (type of sample)
1 acc (M)
2 acc + hom + stabt.0 (TMB), stabt=5, RT (S)
3 acc (M)
4 acc + hom + stabt.0 (TMB), stabt.5, RT (S)

Duplicate samples were analysed
acc=accuracy, hom=homogeneity, stab=stability (hours), T=top, M=middle, B=bottom position of container
S=stability sample taken at middle position of container
RT=room temperature
Duration of treatment / exposure:
(P) Males: 14days before mating. during mating, and up to the day prior to necropsy. Exposure period was at least until the minimum total dosing period
of 28 days was completed. Overall males were exposed for 31 days.
(P) Females: 14 days before mating, up to 18 days during mating, 21 days during resulting pregnancies, at least 3 days through weaning of their F1 offspring. Overall females were exposed for 42 to 45 days.
Frequency of treatment:
Once daily for 7 days per week
Details on study schedule:
- Age at mating of the mated animals in the study: approx. 12 weeks
- Dosing approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 150, 500 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:based on findings from dose-range finding study
- dose volume: 20 ml/kg body weight. Actual dose volumes were calculated according to the latest body weight.



Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily; Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard
arena. Arena observations were not performed when the animals were mating, or housed individually. The time of onset, degree and duration was recorded. All symptoms were recorded and graded according to fixed scales:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe
# Cage debris of pregnant females was examined to detect abortion or premature birth, if applicable. Signs of difficult or prolonged parturition were recorded, if applicable.


BODY WEIGHT: Yes
- Time schedule for examinations: all animals on the first day of exposure and weekly thereafter. Mated females were weighed an Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and on Days 1 and 4 of lactation.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OTHER: Functional Observations
The following tests were performed in 5 males and 5 females, randomly selected from each group:
# hearing ability, pupillary reflex, static righting reflex and grip strength (Score 0 = normal/present, score 1 = abnormal/absent).
# motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).
During the motor activity test, males were caged individually and females were caged with their offspring.
The assigned males were tested during week 4 of treatment and the assigned females were tested during lactation (all before blood sampling).
In order to avoid hypothermia of pups, dams were removed from the pups for not more than 30-40 minutes.

Oestrous cyclicity (parental animals):
estrous cyclicity not examined

- histopathological examination of reproductive organs (cervix, ovaries, uterus, and vagina) of all animals that failed to mate, conceive, sire
or deliver healthy offspring. These animals include:
Group 1 (control): Female 50.
Group 2 (low dose): Females 51, 55 and 60.
Group 4 (high dose): Females 73, 75, 77 and 79.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
- testis weight, epididymis weight, examination of staging of spermatogenesis in slides from testes of five selected animals from control and high dose group
- histopathological examination of reproductive organs from males that failed to mate: i.e. coagulation gland, epididymis, prostate gland, seminal vesicles, testis. These animals include:
Group 1 (control) : Male 10
Group 2 (low dose) : Males 11, 15 and 20
Group 4 (high dose): Males 33, 35, 37 and 39
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups (was determined on day 1 and 4 by assessment of the ano-genital distance), stillbirths, live births, postnatal mortality, presence of gross anomalies (The numbers of live and dead pups at the First Litter Check (= check at Day 1 of lactation) and daily thereafter were determined. If possible, defects or cause of death were evaluated. Animals showing pain, distress or discomfort, which is considered not transient in nature or is likely to become more severe, were sacrificed for humane reasons), weight gain (live pups were weighed on days 1 and 4 of lactation), physical or behavioural abnormalities (detailed clinical observations at least ince daily)

GROSS EXAMINATION OF DEAD PUPS: yes
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, following completion of mating period
- Maternal animals: All surviving animals: females which delivered: on lacttation day 5 or shortly therafter; females which failed to deliver: on post-mating day 24 to 26, females with total litter loss: within 24 hours of litter loss

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cranial, thoracic, and abdominal tissues and organs, with special attention being paid to the reproductive organs

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organ weights (and terminal body weight) was recorded:
From 5 selected animals/sex/group: Adrenal glands, Liver, Brain, Spleen, Epididymides, Testes, Heart, Thymus, Kidneys
From all remaining males: Epididymides, Testes

histopathologic examinations were performed on:
- The preserved organs and tissues of the selected animals of Groups 1(control) and 4 (high dose).
- gender specific investigation (e.g. The additional slides of the testes of the selected 5 males/group of Groups 1 and 4 to examine staging of spermatogenesis, further details see above)
- All gross lesions of all animals (all dose groups)

On detection of possible treatment-related changes in the organs of any animal in the high dose group, histological examination was extended to lungs, thymus, liver, kidneys (all males and females) and adrenals (males only) of five selected animals of Groups 2 and 3.
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at day 4 of lactation.

GROSS NECROPSY and any other examinations
- All offspring was sexed and externally examined if practically possible. The stomach was examined for the presence of milk. Descriptions of all macroscopic abnormalities was recorded. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in neutral phosphate buffered 4% formaldehyde solution, for possible further examination.
Statistics:
The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate were applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied instead of the Dunnett-test if the data could not assumed to follow a normal distribution.
• The Fisher-exact test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the sam printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
Percentage mating = (Females mated / Females paired) * 100
Fertility index = (Females achieving a pregnancy / Females paired) * 100
Conception rate = (Females achieving a pregnancy / Females mated) * 100
Gestation index = (Number of females with living pups / Number of females pregnant) * 100
Duration of gestation: number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Viability index = (number of alive pups on day 4 p.p. / number of pups born alive) * 100
- percentage live males at first litter check: (number of live male pups)/(number of live pups) * 100
- percentage live females at first litter check: (number of live female pups)/(number of live pups) * 100
Percentage of postnatal loss Days 0-4 post partum (p.p.) = (number of dead pups on day 4 p.p. / number of live pups at First litter check) * 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
no animal died during the study, 2 females were killed due to total litter loss; high dose group: Slight to moderate salivation was noted in all males and females treated; incidentally rales were noted in two males
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
high dose group: reduced body weights and body weight gains of male and female animals; food consumption was reduced in females during lactation
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
high dose group: reduced body weights and body weight gains of male and female animals; food consumption was reduced in females during lactation
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
high dose group: decrease in relative number of eosinophils in males and females
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
high dose group females: decrease in gestation index

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- No treatment related mortality occurred during the study period.
- Two females were sacrificed before the end of the study period. One animal treated at 50 mg/kg (Female 60) was sacrificed as the animal was cannibalizing the pups on Day 1 of lactation and one female at 500 mg/kg (Female 79) was sacrificed due to total litter loss on Day 2 of lactation.
This animal had five pups, three pups were found dead on Day 1 of lactation, and two other pups were found dead on Day 2 of lactation.

- high dose group: Slight to moderate salivation was noted in all males and females treated; incidentally rales were noted in two males (Males 37 and 38) and piloerection was noted in one female (Female 79) at the end of treatment.
- all dose groups except control: yellow discolouration of the urine was noted in all animals

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Slightly reduced body weight and body weight gain was noted in males treated at 500 mg/kg during the complete study period (not always statistically significant). Reduced body weight and body weight gain was also noted in females treated at 500 mg/kg on Days 14 to 20 post-coitum and during lactation (not always statistically significant).

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Food consumption before or after allowance for body weight was reduced during lactation in females treated at 500 mg/kg (statistically not significant). Other changes in food consumption were considered to be of no toxicological relevance.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
not examined

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
In males suspected of infertility, there were no findings in the reproductive organs of any of the animals which would account for poor reproductive performance. The spermatogenic staging profiles were normal for all group 1 and group 4 males evaluated.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Of all animals, three females did not mate (one in the control group, one in the low dose group and one in the high dose group) and two females had implantations sites only (high dose group).

ORGAN WEIGHTS (PARENTAL ANIMALS)
- high dose group: Liver/body weight ratlos were increased with statistical significance for males, which correlated with the findings at macroscopic and microscopic examination; decreased epididymidis weight in males
- high dose group: Decreased thymus weight and thymus/body weight ratlos were noted in males and females and correlated in some animals with the atrophy noted at microscopic examination. Furthermore, increased kidney weight and kidney/body weight ratlos were noted in females, which correlated in some animals with the observed basophilia. However, as these microscopic findings were not considered to be related to treatment, the
toxicological relevance of these organ weight changes remains unclear.
- Other changes in organ weight comprised increased heart weight in males at 50, 150 and 500 mg/kg and females at 500 mg/kg (not always statistically significant), increased relative brain weight in males at 500 mg/kg and increased adrenal weight in females at 150 and 500 mg/kg.
- Other changes (increased liver weight in females at 50 mg/kg, decreased testes weight in males at 50 mg/kg) were considered to be of no toxicological relevance in absence of a dose response relationship.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- high dose group: pale discolouration of the liver was noted in five males; many grey-white foci were found an the lungs of 4 females
- One female at 50 mg/kg (Female 60) showed reddish contents in the stomach. This animal was sacrificed due to cannibalism of the pups.
- One female at 50 mg/kg (Female 51) showed an enlarged cervix and uterus. The uterus was filled with fluid. These were signs of pseudo pregnancy and were considered to be unrelated to treatment.
- Incidental findings among control and treated animals are occasionally seen among rats used in these types of study and in the absence of a dose response relationship they were considered changes of no toxicological significance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- In females suspected of infertility, animal 75 (group 4, high dose) had endometrial inflammation. Animals 73 and 77 (group 4) had vaginal epithelial mucification (possible oestrus cycle disturbance). In animals 50 (control), 60 (Group 2, low dose) and 79 (Group 4), there were no findings to account for infertility. Animal 51 (group 2) exhibited endometrial squamous metaplasia and animal 55 (group 2) had a deciduoma. As no similar findings were noted at the mid or high dose group, the toxicological relevance of these findings was doubted.
- Further details on microscopic investigations of parental animals (nor related to reproduction) can be found in section 7.5.1.

OTHER FINDINGS (PARENTAL ANIMALS)
- Details on clinical laboratory investigations of parental animals can be found in section 7.5.1.
- Functional observations: Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
The motor activity test showed an increase in activity at the low sensor for females at 500 mg/kg. No other effects were noted in the motor activity test.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: various findings in high dose group (e.g. reduced body weights and bw gain, haematological and clinical biochemistry findings, macroscopic and microscopic findings in liver, lung and adrenal glands)
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: high dose group: average and total number of living pups per litter was reduced when compared to controls
Remarks on result:
other: Developmental
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: decreased gestation index in high dose group
Remarks on result:
other: Reproduction and fertility

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
high dose group: average and total number of living pups per litter was reduced when compared to controls
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:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
At the first litter check (post natal day 1;PND1), the average and total number of living pups per litter was reduced at 500 mg/kg (average of 6.9 pups per litter) when compared to concurrent controls (average of 16.0 pups per litter).Furthermore, an increased incidence in postnatal loss was noted at 50, 150 and 500 mg/kg, resulting in a reduced viability index. No dose response relationship could be established between the treated groups.

There was an increased incidence in missing and cannibalized pups, correlating with the increased post natal loss noted at 50, 150 and 500 mg/kg when compared to the concurrent controls. The increased incidence in postnatal loss might be caused by possible developmental effects.

BODY WEIGHT (OFFSPRING)
(Mean) body weights were similar for the control and treated groups.

CLINICAL SIGNS; GROSS PATHOLOGY and OTHER FINDINGS (OFFSPRING)
Incidental findings consisted of a small, pale or weak appearance, red spot or discolouration (neck, head, back, tail and/or tail apex), scab (hindleg, nose), wound (nose), blue discolouration abdomen and swelling of the head. Macroscopic examination of the pups revealed no milk in the stomach and a small appearance. No relationship with treatment was established for these observations or they were considered to be within the normal biological variation for rats of this age and strain.

Further details on Reproduction, Fertility and Developmental parameters are presented in the attached document.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In this OECD 422 study in rats the orally administered test material induced changes only in the highest dose tested; i.e. changes in clinical appearance (slight salivation), functional observations (slight hyperactivity), body weights and food consumption (decreased), clinical laboratory investigations decreased eosinophils, ALT and AST activity increased), macroscopic and microscopic examinations (liver, lung and adrenal glands), which correlated with changes in organ weights. There were also effects related to reproduction (decreased gestation index) and litter observation (reduced average and total number of living pups) that were considered to be an effect of treatment.
An increased incidence in postnatal loss (due to increased cannibalism) was noted at 50, 150 and 500 mg/kg, resulting in a reduced viability index. No dose response relationship could be established between the treated groups.
From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for parental and reproduction toxicity of 150 mg/kg bw/day was established for the test item. The study authors indicated that due to the uncertainties coming from the increased number of postnatal losses no developmental NOAEL could be derived. As developmental effects did not follow a clear dose response relationship and a reduced number of pups was observed in the highest dose group a NOAEL of 150 mg/kg/day has been suggested by the registrant for developmental toxicity, resulting in a NOAEL of 150 mg/kg bw/day for parental and reproduction/developmental toxicity for the test item.
Executive summary:

In this guideline study according to OECD TG 422 the test material was administered by daily oral gavage to male and female Wistar rats at dose levels of 0, 50, 150 and 500 mg/kg/day. The males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 31 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 3 days of lactation (for 42 to 45 days). Formulation analysis showed that the formulations were prepared accurately, were homogeneous and were stable for at least 5 hours at room temperature.

No treatment related mortality occurred during the study period.

Slight to moderate salivation was noted in all males and females treated at 500 mg/kg. Furthermore, incidentally rales were noted in two males at 500 mg/kg and piloerection was noted in one female at 500 mg/kg at the end of treatment.

Yellow discolouration of the urine was noted in all animals of Groups 2, 3 and 4 in a dose dependant manner. This could be due to excretion of the test compound or a metabolite in the urine. Without corroborative findings for clinical biochemistry parameters and as no macroscopic or microscopic abnormalities of the kidneys were observed, this finding was not considered to be toxicologically relevant.

At 500 mg/kg, reduced body weight gain was noted in males during the treatment period and in females on Days 14 to 20 post-coitum and during lactation (not always statistically significant). Furthermore, at 500 mg/kg food consumption before or after allowance for body weight was reduced during lactation in females (statistically not significant).

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals, The motor activity test showed an increase in activity at the low sensor for females at 500 mg/kg, which might be due to hyperactivity of the dam and/or pups.

At 500 mg/kg, a treatment related decrease in eosinophils was noted in males and females. Furthermore, treatment related effects were noted in clinical biochemistry (mainly in males). These findings comprised high alanine aminotransferase, aspartate aminotransferase activities (also noted in females at 500 mg/kg) and alkaline phosphatase activities and high cholesterol levels. Cholesterol levels were also increased in males treated at 150 mg/kg, but to a lesser extent. These findings at 500 mg/kg correlated with the macroscopic or microscopic effects on the liver, e.g. pale discolouration, increased liver/body weight ratios and hepatocellular vacuolation of the liver at a minimal or slight degree. In addition, calcium levels were increased in males and females at 500 mg/kg. Besides microscopic changes in the liver, minor treatment related morphological alterations were noted in the lungs and adrenal glands: In the lungs, alveolar macrophage foci were increased in incidence and severity to moderate in females at 500 mg/kg. In the same organ lymphocytic alveolar inflammation was slightly increased in incidence in males and in incidence and severity to moderate in females. These findings correlated with the grey-white foci observed in females at 500 mg/kg. In the adrenal glands of males vacuolation in the zona fasiculata at minor degrees of severity was sbightly increased in incidence at 500 mg/kg which was not statistically significant. However there was a positive trend. The findings in liver, lung and adrenal glands were chiefly minor in nature and may be regarded as either slight increases in spontaneously occurring conditions or adaptive. As such they were considered to be indicators of slight toxicity to the test-item.

The organ weight changes in thymus and kidney correlated with the microscopic findings in these organs, e.g. atrophy and basophilia respectively. No corroborative findings were noted for the changes in weight of the heart, epididymides and brain. These changes were mild in nature and in absence of corroborative findings or a clear dose response relationship, the toxicological relevance of these changes remains unclear.

Findings related to reproductive and developmental toxicity:

The gestation index was decreased in females at 500 mg/kg. No effect was noted an the duration of gestation and precoital time at 50, 150 or 500 mg/kg. Of all animals, three females did not mate (one in the control group, one in the low dose group and one in the high dose group) and two females had implantations sites only (high dose group). In males suspected of infertility, there were no findings in the reproductive organs of any of the animals which would account for poor reproductive performance. Further, the spermatogenic staging profiles were normal for all Group 1 and Group 4 males evaluated. In females suspected of infertility, animal 75 (group 4) had endometrial inflammation. Animals 73 and 77 (group 4) had vaginal epithelial mucification (possible oestrus cycle disturbance).

At 500 mg/kg, the average and total number of living pups per litter was reduced at the first litter check, (average of 6.9 pups per litter) when compared to concurrent controls (average of 16.0 pups per litter). An increased incidence in postnatal loss (due to increased cannibalism) was noted at 50, 150 and 500 mg/kg, resulting in a reduced viability index. No dose response relationship could be established between the treated groups.There was an increased incidence in missing and cannibalized pups, correlating with the increased post natal loss noted at 50, 150 and 500 mg/kg when compared to the concurrent controls. In surviving pups no treatment related changes in developmental indices were noted. Furthermore, (mean) body weights were similar for the control and treated groups.

CONCLUSION

In conclusion, treatment with test material by oral gavage in male and female Wistar rats at dose levels of 0, 50, 150 and 500 mg/kg/day revealed clear parental and reproduction toxicity at 500 mg/kg body weight/day. At the same dose level there was a significant difference in number of living pups at the first litter check compared to controls.

Moreover increased incidence of postnatal loss, cannibalism and/or missing pups was observed at all treated groups. No dose response relationship could be established. No such increased incidence was noted in concurrent and historical control data. However, the cause of the observed cannibalism was unclear and might be related to developmental effects, which could affect the NOAEL.

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for parental and reproduction toxicity for the test item of 150 mg/kg bw/day was established. The study authors indicated that due to the uncertainties coming from the increased postnatal loss no developmental NOAEL could be presented. Taking the fact that no dose response relationship was observed the registrant indicated that a NOAEL for developmental of 150 mg/kg/day which pays tribute to the observed reduced number of pups in the highest dose group could be installed, too.

Overall a No Observed Adverse Effect Level (NOAEL) for parental and reproduction/developmental toxicity for the test item of 150 mg/kg bw/day was established.