Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 25-November 19, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 25- November 19, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hino Breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: 10 weeks
- Weight at study initiation: males 355-394 g and females 240-285 g, females satellite groups 247-279 g
- Fasting period before study:
- Housing: During acclimatization animals were housed individually in stainless stgeel suspended ages. Animals were paired in males' cages. Dams were housed individually in plastic cages floored with an autoclaved substate of wood chips on Day 18 of pregnancy and thereafter. The dams were housed individually in stainless steel suspended cages on Day 4 of lactation and thereafter.
- Diet: ad libitum to feeders of solid diet (CRF-1, Oriental Yeast Co., Ltd.)
- Water: ad libitum tap water
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.9-24.3
- Humidity (%): 44-63
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test substance was measured out and dissolved in corn oil to a concentration of 40 mg/ml. Dosing preparations containing the test substance at concentrations of 10 and 2.5 mg/ml were prepared by serial dilution of the dosing preparation at 40 mg/ml with corn oil. Preparations were used within 4 hours and 54 minutes of preparation. Dosing volumes 5 ml/kg/day on the most recent body weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Concentration in vehicle: same dosing volume.
- Lot/batch no.: V8F7016
Details on mating procedure:
- M/F ratio per cage: 1/1 one female was cohabitated with one male of the same treatment group, avoiding sibling mating
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal smear, or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 of preganancy. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually and was allowed to deliver the litters spontaneously and observed for delivery conditions and completion of delivery daily from Day 21 of pregnancy to day 25 of pregnancy. The day when deliver was completed was defined as day 0 of lactation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of the substance in dosing preparations which were used for both sexes in the test groups on the first day of dosing and for the males in the test gorups on the last dosing day were measured by gas chromatography. Concentrations were between 92.9 and 95.1% of target concentration.
Duration of treatment / exposure:
Males treated for 14 days before mating and thereafter 28 days, total of 42 days. Half of the males per group were given a 14-day recovery period.
Females treated for 14 days before mating, during the mating period (max 4 days), and during pregnancy up to Day 6 of lactaction, a total of 44 - 48 days. Females of satellite groups, treated for 42 days, were given a 14-day recovery period.
Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
Daily
Details on study schedule:
- Dose selection rationale: Dose levels based on a preliminary study in which death was noted at 500 mg/kg/day and higher. The dosing length in the present study was to be more than 3 times longes than in the preliminary study, the high dose was selected to be 200 mg/kg/day, at which toxicity was expected to occur and lower dose levels were calculated using a common ration of 4.
- Rationale for animal assignment (if not random): Random sampling method so that the mean body weight and variance were as equal as possible among the groups.
- Post-exposure recovery period in satellite groups: 14 days.
Dose / conc.:
12.5 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 males including 6 with a recovery period.
12 females
6 females in satellite group with recovery period.
Control animals:
yes, concurrent vehicle
Positive control:
not applicable
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Animals were observed for mortality and clinical signs twice a day, once before and once after administration, once a day during the recovery period and once on the day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week and on the day of necropsy, with females during pregnancy on Days 0, 7, 14 and 21 and on Days 0, 4 and 6 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Time schedule: approx twice a week

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: approx twice a week

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After last dosing day and after temination of recovery period.
- Anaesthetic used for blood collection: Yes, by ip injection of sodium pentobarbital
- Parameters examined: according to OECD 422 (1996)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see haematology
- Parameters examined: according to OECD 422 (1996)

URINALYSIS: Yes
- Time schedule for collection of urine: Fresh urine and 24-hour urine was collected. For males on day 37 of administration and day 9 of recovery, for females on day 5 of lactation and day 9 of recovery.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: All animals were observed for FOB before start of administration and once a week during administration.
- Dose groups that were examined: all dose groups
- Battery of functions tested: FOB consisted of observations on: posture, palpebral closure, biting behavior and convulsions; ease of removal from cage, ease of handling, muscle tone, fur conditions, lacrimation, salivation, and respiration. In the open field, frequency of rearing and frequency of grooming for 2 minutes, gait, palpebral closure, consciousness, behavioral abnormalities, and righting reflex.
Sensory reactivity and grip strength in males before necropsy, in 6 females/group on Day 3 of lactation. Spontaneous Motor Activity in males on day 40 of administration and females on day 4 of lactation.

OTHER:
Mating: After 14 days of treatment, males and females were paired continuously for a maximum period of 14 days until copulation was confirmed.
Nursing conditions: Dams were observed for nursing conditions once a day from days 0 to 4 of lactation.
Oestrous cyclicity (parental animals):
Estrous cycles: Females were observed once a day from the first dosing day to the day before confirmation of copulation.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight, microscopic examinations of testes, epididymides, seminal vesicles, prostate.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: the number of pups born, their sexes, number of stillbirths, live pups born, and external abnormalities.
- Mortality: once a day
- Clinical signs: once a day
- Body weights: Live pups were weighed on Days 0 and 4 of lactation.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
Organ weights in males: brain, pituitary, salivary glands, thyroids, thymus, heart, liver, spleen, kidneys, adrenals, testes and epididymides.
Females: number of corpora lutea and number of implantation sites were counted. After necropsy organ weights: same as males + ovaries and uterus.
HISTOPATHOLOGY: Yes
Organs and tissues from males and females of the high dose and control group were examined. When abnormalities were found, e.g. stomach, lower doses were also examined. Organs and tissues examined: heart, lungs, trachea, liver, pancreas, salivary glands, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, thymus, spleen, lymph nodes, kidneys, urinary bladder, testes, epididymides, seminal vesicles, prostate, pituitary, adrenals, thyroids, parathyroid glands, brain, spinal cord, sciatic nerves, eyeballs, Harderian glands, bone marrow and bones, testes, ovaries, uterus, vagina, and mammary glands.
Statistics:
Data were tested by Bartlett's test for homogeneity of variance, when variances were homogeneous, Dunnett's test was performed to compare the control group with each of the groups treated with substance. When variance was heterogeneous, Steel's test was performed.
FOB and reproduction/developmental parameters data were tested by Steel's test. Copulation index, fertility index and gestation index were tested by Fisher's exact test.
Effects in stomach: Steel's test was performed. When significant differences were seen, dose-responsiveness was tested by Cochran-Armitage exact test.
Reproductive indices:
For each group, the following calculations were performed:
- Copulation index: Number of animals which copulated/Number of animals paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Gestation index: Number of dams with live pups/Number of pregnant females x 100
Offspring viability indices:
- Implantation index: number of implantation sites/number of corpora lutea x 100
- Delivery index: number of pups born/number of implantation sites x 100
- Birth index: Number of live pups born on Day 0 of lactation/Number of implantation sites x 100
- Live birth index: Number of live pups born on Day 0 of lactation/Number of pups born x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100
- Incidence of external abnromalities: number of pups with external abnormalities/number of live pups born x 100
- Sex ratio: number of males/number of females
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation was noted in 200 and 50 mg/kg bw/day group animals after administration. This was considered to be due to the irritancy of the substance and since no further neurological effects were found, not regarded as a systemic effect. During recovery no clinical signs in females and only one male with soiled perineal area was noted.
Mortality:
no mortality observed
Description (incidence):
No dead or moribund males or females were noted in any group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight was significantly lower in the males at 200 mg/kg bw/day throughout the administration period and in the females at the same dose level on Day 15 of administration, throughout pregnancy, and on Days 0 and 6 of lactation. Body weight was also lower in the males at 50 mg/kg/day on Days 29 and 36 of administration. However, these findings at 50 mg/kg bw/day are not considered attributable to the test substance, since (1) the differences from the control group were small, (2) these findings were transient changes, and (3) food consumption was not adversely affected by the test substance at this dose level. During the recovery period, body weight stayed lower in both sexes at 200 mg/kg bw/day but tended to revert to the normal body weight.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the measurement of food consumption, lower food consumption was noted in the males at 200 mg/kg bw/day at the beginning of the administration period, but no changes attributable to the test substance were noted in the females at this dose level. During the recovery period, food consumption was higher in the males at 200 mg/kg/day, but this finding is considered to be a recoverable change which occurred after termination of the administration period.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
In the measurement of water consumption, higher water consumption was noted in the males at 200 mg/kg bw/day at the end of the administration period, in the females at this dose level before the start of mating, and in the females at 200 and 50 mg/kg/day during the lactation period. However, these findings were not regarded as having been caused by the toxicity of the test substance, since all of them were transient changes. During the recovery period, water consumption was higher in the males at 200 mg/kg bw/day, but no changes attributable to the test substance were noted in the females at this dose level.
Haematological findings:
no effects observed
Description (incidence and severity):
No changes attributable to test substance were noted in either sex on termination of the administration period or recovery period.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No changes attributable to test substance were noted in either sex on termination of the administration period or recovery period.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No changes attributable to test substance were noted in either sex on termination of the administration period or recovery period.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
In the measurement of spontaneous motor activity in the females at 200 mg/kg bw/day, the ambulatory count and vertical count were lower 70 minutes after administration, and a lower total vertical count and a tendency for total ambulatory count to be lower were noted on termination of the administration period. In the males, no changes attributable to the test substance were noted in the spontaneous motor activity. Regarding FOB, sensory reactivity, and grip strength, no changes attributable to the test substance were noted in either sex at any dose level.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the organ weight measurement on termination of the administration period, a higher relative spleen weight and a tendency for the absolute spleen weight to be higher were noted in the males at 200 mg/kg bw/day, and higher absolute and relative spleen weights were noted in the females at this dose level. On termination of the recovery period, no changes attributable to the test substance were noted in either sex at any dose level. However, the histopathological examinations on termination of the administration period (see below) shows that there were no changes attributable to the test substance in the spleen in either sex, and hematological examinations shows that the parameters related to the organ weight changes were not affected by the test substance. Therefore, the findings in the spleen weights at 200 mg/kg bw/day in the present study are considered to be minor changes.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-like residuals were noted in the stomach of animals of both sexes at 200 mg/kg bw/day.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the histopathological examinations, the following findings were noted in the males:
slight or mild hyperplasia of squamous epithelium in the forestomach in all 6 males at 200 mg/kg bw/day and in 1 male at 50 mg/kg bw/day, slight or mild hyperkeratosis in the forestomach in all 6 males at 200 mg/kg bw/day, slight inflammatory cell infiltration in the forestomach in 1 male at 200 mg/kg bw/day, and cystic changes in the epithelial layer of the forestomach in 1 male at 200 mg/kg bw/day. In the females at 200 mg/kg bw/day, the following findings were noted: slight hyperplasia of squamous epithelium in the forestomach in 3 females, slight inflammatory cell infiltration in the forestomach in 1 female, and cystic changes in the epithelial layer of the forestomach in 1 female. From these findings in the stomach, it can be said that the above-mentioned higher water consumption in both sexes at 200 mg/kg bw/day was a change to alleviate the irritancy of m-xylylene diisocyanate given to the stomach; the higher water consumption noted in the present study is not judged to be a toxicological change. Since no histopathological changes in the stomach were noted in either sex on termination of the recovery period, higher water consumption occurring during administration will revert to normal water consumption after the discontinuation of administration.
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
In the examinations for reproductive/developmental toxicity in parent animals, no changes attributable to the test substance were noted in the number of estrous cases during the administration period (for 14 days) before the start of mating, copulation index, number of conceiving days, number of pregnant females, fertility index, length of gestation, gestation index, number of corpora lutea, number of implantation sites, implantation index, delivery conditions, or nursing conditions.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Decreased body weight in males and pregnant females.
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
In the pups (F1), no changes attributable to the test substance were noted in the number of pups born, number of stillbirths, number of live pups born on Day 0 of lactation, delivery index, birth index, live birth index, sex ratio, number of live pups on Day 4 of lactation, viability index on Day 4 of lactation, clinical signs, body weight on Day 0 or 4 of lactation, appearance, or necropsy findings.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Based on the results of a repeated dose study with screening for reproductive and developmental effects, the reproductive/developmental NOAEL is sconcluded to exceed 200 mg/kg bw/day as no effects were noted in any parameter in either sex. At 200 mg/kg bw/day, a decreased body weight was found in males and pregnant females and thus the parental NOAEL is set at 50 mg/kg bw/day.
Executive summary:

Rats were administered 0, 12.5, 50 or 200 mg/kg bw/d of test substance in a study performed according to OECD 422 (1996). Rats were dosed from 14 days before mating during mating and afterwards (total 42 days) for males and during mating period, pregnancy and up to lactation day 6 for females. No mortality occurred. Salivation was noted at 50 and 200 mg/kg bw/d in both sexes. Body weight was decreased in both sexes at 200 mg/kg bw/d. For food and water consumption only transient effects were noted which may be due to irritation of the stomach. No test substance-related effect was seen on haematological, clinical chemistry and urinalysis parameters or at gross pathology. Histopathology revealed an increased incidence of hyperplasia of squamous epithelium and hyperkeratosis in the forestomach in both sexes at 200 mg/kg bw/d. Inflammatory cell infiltration in the forestomach and mild cystic changes in the epithelial cell layer of the forestomach were noted in one male and one female at 200 mg/kg bw/d. In the pups (F1), no changes attributable to the test substance were noted in the number of pups born, number of stillbirths, number of live pups born on Day 0 of lactation, delivery index, birth index, live birth index, sex ratio, number of live pups on Day 4 of lactation, viability index on Day 4 of lactation, clinical signs, body weight on Day 0 or 4 of lactation, appearance, or necropsy findings. In addition, no effects on reproduction organs in parental animals were observed.

Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 28, 1990 - August 12, 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study meets the standards of the EC-method and was conducted according to GLP principles. However, deviations from the test guideline were noted, which affect the reliability of the study.
Qualifier:
according to
Guideline:
other: The Japanese Guidelines for Screening Toxicity Testings of Chemicals (1986)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
no full histopathology was done
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: At the start of the exposure, the rats are 5 weeks old.
- Weight at study initiation: males: 157-188, females: 123-151
- Housing: Two rats of the same sex were housed in a polycarbonate cage with hard wood chip bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5-7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25 °C
- Humidity (%): 40-70%
- Air changes (per hr): 12 changes/hour
- Photoperiod (hrs dark / hrs light): 12/12 hours
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance solutions were prepared by dissolving the test substance in the vehicle (olive oil) at desired concentrations using an ultra high speed homogenizer. The test substance solutions were prepared once a week and stored in the refridgerator until just before use. The stability of the test substance solutions for 7 days after preparation was confirmed by chemical analysis.

VEHICLE
- Amount of vehicle (if gavage): 1.0 ml per 100 g body weight in all rats
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance solutions for 7 days after preparation was confirmed by chemical analysis. No results included in the report.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once a day in the morning
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Main study:
0 and 500 mg/kg bw/day : 12 (6 for 14-day recovery)
15 and 100 mg/kg bw/day: 6
Additional study:
0, 2, 10 mg/kg bw/day: 6 females
2 mg/kg bw/day: 6 female
10 mg/kg bw/day: 6 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A 1-week preliminary study was performed at the doses of 60, 500 and 700 mg/kg bw/day. It was found that the substance produced toxic effects on animals of the 500 and 700 mg/kg bw/day dose groups. Suppressed body weight gains, thickening of the forestomach and decreased spleen weight.
- Rationale for animal assignment (if not random): so that the body weights in each group became almost the same
- Rationale for selecting satellite groups: to evaluate recovery. Satelite animals in the control and the top dose group
- Post-exposure recovery period in satellite groups: 14 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily, and with palpation once a week

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each cage (two animals) determined and mean daily diet consumption calculated as g food/animal/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data:No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: just before necropsy
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: No data
- How many animals: all
- Parameters were examined: erytrocyte count, Leucocyte count, platelet count, hemoglobin concentration, hematocrit, leucocyte count differential, reticulocyte count, prothrombin time, activated partial thromboplastin time, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: just before necropsy
- Animals fasted: No data
- How many animals: all
- Parameters were examined: total protein, albumin, A/G ratio, Glucose, triglyceride, total cholesterol, urea nitrogen, creatinine, calcium, inorganic phosphorus, GOT(AST), GTP(ALT, gamma-GTP, ALP, sodium, potassium, chloride.

URINALYSIS: Yes
- Time schedule for collection of urine: two days before necropsy
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters were examined: pH, occult blood, protein, glucose, ketone bodies, bilirubin, urobilinogen

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes , brain, pituitary, eyes(with Hardian glands), lungs, stomach, thyroids (with parathyroids), heart, liver, spleen, kidneys, adrenals, urinary bladder and bone marrow (femur), testes or ovaries. Of all organs removed at the end of treatment, the heart, liver, kidneys, adrenals, spleen and stomach from rats of both sexes of the control and high dose group and the kidneys from 1 male rat of the low dose group, were examined microscopically. The OECD guideline states that full histopathology should be done.
Statistics:
Barlett's test.
Anova and dunnett's multiplerange test or Kruskal-Wallis and Dunnet's type rank sum test.
Armitage's chi2test was used for urunary paper tests
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Almost all rats of both sexes of the 500 mg/kg bw/day dose group had salivation after administration continuously after 5 days of treatment (reversible). Although related to the substance, it was only slight and readily improved, disappearing after discontinuation. Some male rats also had soft faeces, reduced spontaneous movement and rales. These symptoms were transient and seemed to be slight.
Mortality:
no mortality observed
Description (incidence):
No death occurred during the periods of treatment and recovery.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gains were suppressed in male rats of the 500 mg/kg bw/day dose group during treatment and recovery (up to 13% on day 7 and day 28).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Decreased in rats of both sexes of the high dose group early in treatment, returned to normal or tended to increase thereafter, and thus considered only slight.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Starting at the 15 mg/kg bw/day dose group, the hematocrit values decreased in the female animals:
At 15 mg/kg bw/day: 5% decrease compared to control
At 100 mg/kg bw/day: 4.8% decrease compared to control
At 500 mg/kg bw/day: 6.4% decrease compared to control
These changes, although statistically significant, are not considered toxicologically relevant, since the decrease is not dose-dependent and only minor. The author of the report considers the effects to be related to the substance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At highest dose, increase in GPT in rats of both sexes, increase in GOT in males, decrease in glucose and increase in A/G ratio in female rats.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Presence of occult blood in male rats of high dose group and decreased protein in female rats of the high dose dose group. These effecst were not considered to be toxicologically relevant.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant observations:
Female rats of 500 mg/kg bw/day dose group showed an increase of absolute (12%) and relative (7%) liver weights as well as a reduction in relative (14%) and absolute (18%) ovary weight, and decreased relative and absolute adrenal weight. The latter was also seen in the low dose groups (small change and not dose related). Male rats of 500 mg/kg bw/day dose group showed a statistically significant but very slight decrease in absolute liver weight only (2%).
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In female rats of the high dose group, deposition of glycogen in the liver (6/6) was seen.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on an increase of liver weights and a reduction in ovary weights in the females and a suppression of body weight gain in males at 500 mg/kg bw/day.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
female reproductive system
Organ:
ovary
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

In the additional study with 0, 2 and 10 mg/kg bw/day dose groups, no mortality, no clinical signs, no effect on bodyweight and food consumption, neither on haematological parameters, organ weight and macroscopic examination were observed.

Based on the main study, the effects on organ weights at the highest dose tested and the decrease in body weight gain are considered toxicologically relevant. As all other effects, clinical signs, haematological and biochemical effects, are considered not relevant, the NOAEL is considered to be 100 mg/kg bw/day. Note: no microscopic examination of several organs was performed, therefore the organ weight effects although sometimes slight, are considered relevant.

Conclusions:
In a sub-acute oral study in rats a NOAEL (systemic effects) of 100 mg/kg bw/day was established based on an increase of liver weights and a reduction in ovary weights in the females and a suppression of body weight gain in males at 500 mg/kg bw/day.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Organisation of Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, March 1996
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): m-xylylene diisocyanate, XDI, CAS no. 3634-83-1
- Physical state: colorless clear liquid

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hino Breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: 10 weeks
- Weight at study initiation: males 355-394 g and females 240-285 g, females satellite groups 247-279 g
- Fasting period before study:
- Housing: During acclimatization animals were housed individually in stainless stgeel suspended ages. Animals were paired in males' cages. Dams were housed individually in plastic cages floored with an autoclaved substate of wood chips on Day 18 of pregnancy and thereafter. The dams were housed individually in stainless steel suspended cages on Day 4 of lactation and thereafter.
- Diet: ad libitum to feeders of solid diet (CRF-1, Oriental Yeast Co., Ltd.)
- Water: ad libitum tap water
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.9-24.3
- Humidity (%): 44-63
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test substance was measured out and dissolved in corn oil to a concentration of 40 mg/ml. Dosing preparations containing the test substance at concentrations of 10 and 2.5 mg/ml were prepared by serial dilution of the dosing preparation at 40 mg/ml with corn oil. Preparations were used within 4 hours and 54 minutes of preparation. Dosing volumes 5 ml/kg/day on the most recent body weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Concentration in vehicle: same dosing volume.
- Lot/batch no.: V8F7016
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of the substance in dosing preparations which were used for both sexes in the test groups on the first day of dosing and for the males in the test gorups on the last dosing day were measured by gas chromatography. Concentrations were between 92.9 and 95.1% of target concentration.
Details on mating procedure:
- M/F ratio per cage: 1/1 one female was cohabitated with one male of the same treatment group, avoiding sibling mating
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal smear, or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 of preganancy. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually and was allowed to deliver the litters spontaneously and observed for delivery conditions and completion of delivery daily from Day 21 of pregnancy to day 25 of pregnancy. The day when deliver was completed was defined as day 0 of lactation.
Duration of treatment / exposure:
Males treated for 14 days before mating and thereafter 28 days, total of 42 days. Half of the males per group were given a 14-day recovery period. Females treated for 14 days before mating, during the mating period (max 4 days), and during pregnancy up to Day 6 of lactaction, a total of 44 - 48 days. Females of satellite groups, treated for 42 days, were given a 14-day recovery period.
Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
12.5 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 males including 6 with a recovery period.
12 females
6 females in satellite group with recovery period.
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Animals were observed for mortality and clinical signs twice a day, once before and once after administration, once a day during the recovery period and once on the day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week and on the day of necropsy, with females during pregnancy on Days 0, 7, 14 and 21 and on Days 0, 4 and 6 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Time schedule: approx twice a week

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: approx twice a week

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After last dosing day and after temination of recovery period.
- Anaesthetic used for blood collection: Yes, by ip injection of sodium pentobarbital
- Parameters examined: according to OECD 422 (1996)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see haematology
- Parameters examined: according to OECD 422 (1996)

URINALYSIS: Yes
- Time schedule for collection of urine: Fresh urine and 24-hour urine was collected. For males on day 37 of administration and day 9 of recovery, for females on day 5 of lactation and day 9 of recovery.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: All animals were observed for FOB before start of administration and once a week during administration.
- Dose groups that were examined: all dose groups
- Battery of functions tested: FOB consisted of observations on: posture, palpebral closure, biting behavior and convulsions; ease of removal from cage, ease of handling, muscle tone, fur conditions, lacrimation, salivation, and respiration. In the open field, frequency of rearing and frequency of grooming for 2 minutes, gait, palpebral closure, consciousness, behavioral abnormalities, and righting reflex.
Sensory reactivity and grip strength in males before necropsy, in 6 females/group on Day 3 of lactation. Spontaneous Motor Activity in males on day 40 of administration and females on day 4 of lactation.

OTHER:
Mating: After 14 days of treatment, males and females were paired continuously for a maximum period of 14 days until copulation was confirmed.
Nursing conditions: Dams were observed for nursing conditions once a day from days 0 to 4 of lactation.

GROSS PATHOLOGY: Yes
Organ weights in males: brain, pituitary, salivary glands, thyroids, thymus, heart, liver, spleen, kidneys, adrenals, testes and epididymides.
Females: number of corpora lutea and number of implantation sites were counted. After necropsy organ weights: same as males + ovaries and uterus.
HISTOPATHOLOGY: Yes
Organs and tissues from males and females of the high dose and control group were examined. When abnormalities were found, e.g. stomach, lower doses were also examined. Organs and tissues examined: heart, lungs, trachea, liver, pancreas, salivary glands, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, thymus, spleen, lymph nodes, kidneys, urinary bladder, testes, epididymides, seminal vesicles, prostate, pituitary, adrenals, thyroids, parathyroid glands, brain, spinal cord, sciatic nerves, eyeballs, Harderian glands, bone marrow and bones, testes, ovaries, uterus, vagina, and mammary glands.
Ovaries and uterine content:
The ovaries, uterus, vagina and mammary glands were examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
- Number of stillbirths: Yes
Fetal examinations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: the number of pups born, their sexes, number of stillbirths, live pups born, and external abnormalities.
- Mortality: once a day
- Clinical signs: once a day
- Body weights: Live pups were weighed on Days 0 and 4 of lactation.
Statistics:
Data were tested by Bartlett's test for homogeneity of variance, when variances were homogeneous, Dunnett's test was performed to compare the control group with each of the groups treated with substance. When variance was heterogeneous, Steel's test was performed.
FOB and reproduction/developmental parameters data were tested by Steel's test. Copulation index, fertility index and gestation index were tested by Fisher's exact test.
Effects in stomach: Steel's test was performed. When significant differences were seen, dose-responsiveness was tested by Cochran-Armitage exact test.
Indices:
- Implantation index: number of implantation sites/number of corpora lutea x 100
- Delivery index: number of pups born/number of implantation sites x 100
- Birth index: Number of live pups born on Day 0 of lactation/Number of implantation sites x 100
- Live birth index: Number of live pups born on Day 0 of lactation/Number of pups born x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100
- Incidence of external abnromalities: number of pups with external abnormalities/number of live pups born x 100
- Sex ratio: number of males/number of females

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation was noted in 200 and 50 mg/kg bw/day group animals after administration. This was considered to be due to the irritancy of the substance and since no further neurological effects were found, not regarded as a systemic effect. During recovery no clinical signs in females and only one male with soiled perineal area was noted.
Mortality:
no mortality observed
Description (incidence):
No dead or moribund males/females were found in any group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight was significantly lower in the females at the high dose level on Day 15 of administration, throughout pregnancy, and on Days 0 and 6 of lactation. During the recovery period, body weight stayed lower at 200 mg/kg bw/day but tended to revert to the normal body weight.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effects on food consumption were found in exposed females.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
In the measurement of water consumption, higher water consumption was noted in the females at this dose level before the start of mating, and in the females at 200 and 50 mg/kg bw/day during the lactation period. However, these findings were not regarded as having been caused by the toxicity of the test substance, since all of them were transient changes. During the recovery period, no changes attributable to the test substance were noted in the females at this dose level.
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Behaviour (functional findings):
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-like residuals were noted in the stomach of animals at 200 mg/kg bw/day.
Neuropathological findings:
effects observed, treatment-related
Description (incidence and severity):
In the histopathological examinations, the following findings were noted in the females at 200 mg/kg/day, the following findings were noted: slight hyperplasia of squamous epithelium in the forestomach in 3 females, slight inflammatory cell infiltration in the forestomach in 1 female, and cystic changes in the epithelial layer of the forestomach in 1 female. From these findings in the stomach, it can be said that the above-mentioned higher water consumption in both sexes at 200 mg/kg/day was a change to alleviate the irritancy of m-xylylene diisocyanate given to the stomach; the higher water consumption noted in the present study is not judged to be a toxicological change. Since no histopathological changes in the stomach were noted in either sex on termination of the recovery period, higher water consumption occurring during administration will revert to normal water consumption after the discontinuation of administration.

Maternal developmental toxicity

Details on maternal toxic effects:
In the examinations for reproductive/developmental toxicity in parent animals, no changes attributable to the test substance were noted in the number of estrous cases during the administration period (for 14 days) before the start of mating, copulation index, number of conceiving days, number of pregnant females, fertility index, length of gestation, gestation index, number of corpora lutea, number of implantation sites, implantation index, delivery conditions, or nursing conditions.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: Based on decreased body weight of pregnant females.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
In the pups (F1), no changes attributable to the test substance were noted in the number of pups born, number of stillbirths or number of live pups born on Day 0 of lactation
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
Only examined until PND 4.
External malformations:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.

In the pups (F1), no changes attributable to the test substance were noted in the number of pups born, number of stillbirths, number of live pups born on Day 0 of lactation, delivery index, birth index, live birth index, sex ratio, number of live pups on Day 4 of lactation, viability index on Day 4 of lactation, clinical signs, body weight on Day 0 or 4 of lactation, appearance, or necropsy findings.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects seen up to and including the highest dose tested.

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of a repeated dose study with screening for reproduction and developmental effects, the reproductive/developmental NOAEL is considered to exceed 200 mg/kg bw/day as no effects were noted in any parameter in either sex. At 200 mg/kg bw/day a decreased body weight was found in pregnant females and thus the parental NOAEL is set at 50 mg/kg bw/day.
Executive summary:

Rats were administered 0, 12.5, 50 or 200 mg/kg bw/d of test substance in a study performed according to OECD 422 (1996). Rats were dosed from 14 days before mating during mating and afterwards (total 42 days) for males and during mating period, pregnancy and up to lactation day 6 for females. No mortality occurred. Salivation was noted at 50 and 200 mg/kg bw/d in both sexes. Body weight was decreased in both sexes at 200 mg/kg bw/d. For food and water consumption only transient effects were noted which may be due to irritation of the stomach. No test substance-related effect was seen on haematological, clinical chemistry and urinalysis parameters or at gross pathology. Histopathology revealed an increased incidence of hyperplasia of squamous epithelium and hyperkeratosis in the forestomach in both sexes at 200 mg/kg bw/d. Inflammatory cell infiltration in the forestomach and mild cystic changes in the epithelial cell layer of the forestomach were noted in one male and one female at 200 mg/kg bw/d. In the pups (F1), no changes attributable to the test substance were noted in the number of pups born, number of stillbirths, number of live pups born on Day 0 of lactation, delivery index, birth index, live birth index, sex ratio, number of live pups on Day 4 of lactation, viability index on Day 4 of lactation, clinical signs, body weight on Day 0 or 4 of lactation, appearance, or necropsy findings. In addition, no effects on reproduction organs in parental animals were observed. The NOAEL for systemic parental effects is 50 mg/kg bw/d based on

decreased body weight and salivation and the NOAEL for developmental toxicity is >= 200 mg/kg bw/d.