1,3-bis(isocyanatomethyl)benzene

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on the results of a reproduction screening study, performed according to OECD guidelines and GLP principles, the reproductive NOAEL was concluded to exceed 200 mg/kg bw/day, as no effects were noted on any relevant parameter in either sex. The parental NOAEL was found to be 50 mg/kg bw/day, based on a decreased body weight in males and pregnant females at 200 mg/kg bw/day.

Link to relevant study records

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 for cross-reference:

reference to same study

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:

other: Crl:CD (SD)

Sex:

male/female

Details on test animals or test system 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/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 bw/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/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 bw/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 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, 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

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:

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: oestrous cycle:

no effects observed

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.

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:

The reproductive/developmental NOAEL can be set at >= 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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

A reliable study is available (Klimisch score 1).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In an OECD 422 study, rats were exposed orally to 0, 12.5, 50 and 200 mg/kg bw/day of XDI. 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 parental NOAEL is 50 mg/kg bw/d and the developmental/fertility NOAEL is >= 200 mg/kg bw/d.

Effects on developmental toxicity

Description of key information

In an oral screening study with rats, no effects on development were observed up to 200 mg/kg bw/day, while maternal toxic effects (decreased body weight) were seen at 200 mg/kg bw/day. These conclusions were confirmed in a prenatal toxicity study, Based on the results of this study, performed according to OECD guideline 414 and GLP principles, the maternal NOAEL was 100 mg/kg bw/day and the developmental NOAEL was at least 400 mg/kg bw/day, the highest dose tested.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Remarks:

Screening study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

June 25-November 19, 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

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

Species:

rat

Strain:

other: Crl:CD (SD)

Details on test animals or test system 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

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:

Daily

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

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

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 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 bw/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/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 bw/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 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, 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.

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.

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 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:

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.

Dose descriptor:

NOAEL

Effect level:

>= 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: no developmental toxicity at the highest dose tested

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: decreased body weight of pregnant females at 200 mg/kg bw/day

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.

Details on embryotoxic / teratogenic effects:
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

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No developmental effects including highest dose tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

The reproductive/developmental NOAEL can be set at >= 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 maternal 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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 24 to August 25, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was initiated after final decision CCH-D-2114489560-42-01/F was received, in which ECHA requested to submit information on a pre-natal developmental toxicity study (Annex IX, Section 8.7.2.; test method: OECD TG 414) in a first species (rat or rabbit), oral route with the registered substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD) [SPF]

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 11 weeks
- Weight at study initiation: 234.5 to 307.2 g.
- Fasting period before study: No
- Housing: individual housing in steel mesh cages (W29.1 × D 26.3 × H 18.0 cm) hung on a water flushing breeding rack (Toyo-Riko)
- Diet: pellet diet CRF-1 (Oriental Yeast), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days (following a quarantine period of 7 days)
The test facility obtained certificates of analyses on the contaminant levels for each lot of pellet diet, and confirmed that the levels were within the acceptable limits proposed by the Japan Experimental Animal Feed Association. The drinking water was analyzed for quality in April 2020 at another inspection agency, in accordance with the specifications of the Water Works Law. The drinking water was analyzed for bacteria (common bacteria and Escherichia coli) in May and June 2020 by the BSRC. The results of these analyses indicated that the levels of contaminants were within the acceptable ranges in the Tap Water Quality Standard.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.9 to 23.0
- Humidity (%): 43 to 56%
- Air changes (per hr): 12 times or more
- Photoperiod (hrs dark / hrs light):12/12 (light on: 7:00, light off: 19:00)

IN-LIFE DATES: From: May 18, 2020 To: June 9, 2020

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was mixed with corn oil and it was administered to the animals once a day by gavage using a stomach tube and a disposable syringe.
At first, 100 mg/mL solution was prepared. A required amount of the test substance was accurately weighed into a measuring cylinder and mixed with an adequate amount of the vehicle. Then the vehicle was added up to the final volume. Dosing formulations of 50 and 25 mg/mL were prepared by diluting 100 mg/mL solution. The prepared dosing formulations were divided for daily use, and stored in the refrigerator until use [actual temperature: 4.0 to 8.1°C, duration of storage: from May 19, 2020 to June 8, 2020 (from the second day of preparation of dosing formulations to the final day of dosing)].

VEHICLE
- Justification for use and choice of vehicle: The substance is highly reactive in water, therefore no aqueous solution could be used.
- Concentration in vehicle: 25, 50 or 100 mg/mL
- Amount of vehicle: 4 mL/ kg bw
- Supplier and Lot no.: FUJI Film Wako Pure Chemical Corporation, Lot No. CAR2149

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability/homogeneity analysis: The 25 and 100 mg/mL dosing formulations were analyzed just after preparation, after 3 days of storage in a tight light-resistant container under refrigeration (acceptable range: 1 to 9°C) and subsequent 6 hours of storage in a tight light-resistant container at room temperature (acceptable range: 1 to 30°C). Remaining (ratio of each mean measured concentration after storage to those just after preparation) and relative standard deviation (RSD) were calculated from the measured concentration. When the remaining are within 100.0±10.0%, the test substance formulations would be judged to be stable. When the RSD are 5.0% or less, the test substance formulations would be judged to be homogeneous.

Concentration/homogeneity analysis: Concentrations of XDI in the dosing formulations were examined at the first and last preparations by the method established previously (Validation of Determination method of m-Xylylene diisocyanate in corn oil: Exp. No. J078 (575-012)). Whether each formulation have been appropriately prepared or not was judged according to the following criteria. The dosing formulations on the first preparation were prepared for concentration/homogeneity analysis, these formulations were not used for dosing.
Acceptance citerium vehicle: No interference peak should be detected at the retention time of XDI on the chromatogram. If the peak is detected, the peak area should be less than 10.0% of that of the standard solution for calibration curve (nominal concentration: 0.005 mg/mL).
Acceptence criteria test item formulations: The ratio to the nominal concentration should be within 100.0±10.0%. The relative standard deviation of the homogeneity analyses should be 5.0% or less.

Details on mating procedure:

Males and females were cohabited on one-on-one basis overnight until evidence of mating is confirmed. For female animals the day when sperm was confirmed in the vaginal smear at examination in the morning time was designated as Day 0 of gestation. Estrus cycles of all animals were examined for at least 7 days prior to mating, and those animals in a suitable condition for mating were subjected to the mating process.
Successfully copulated females were assigned to the test groups on the day of copulation using the system package software for safety study (LATOX-F/V5, FFC), on the basis of the body weight on Day 0 of gestation. Animals copulated on the same day were uniformaly assigned to the groups. The clinical signs were recorded daily during the mating period.

Duration of treatment / exposure:

From Day 5 to 19 of gestation

Frequency of treatment:

Once daily

Duration of test:

16 days

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low dose group

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

Mid dose group

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Remarks:

High dose group

No. of animals per sex per dose:

23 copulated females

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
In a dose range finding study for prenatal developmental toxicity study of m-xylylene diisocyanate in rats (dose levels: 0, 100, 300, 500 mg/kg bw/day, N=5), no dead dams were observed in any groups. In the 300 and 500 mg/kg bw/day groups, decreased food consumption values were observed from Day 5 of gestation (start dosing day) to Day 11 of gestation. Moreover, in the 300 and 500 mg/kg bw/day groups, white patches on the kidneys were observed at gross pathology. However, there were no effects on fetuses in any of the groups. The effects of test substance treatment on dams were slightly in the 300 mg/kg bw/day group, and more severe in the 500 mg/kg bw/day group.
From these results, the 400 mg/kg bw/day (intermediate dose between 300 and 500 mg/kg bw/day) was set as the high dose level, and 200 and 100 mg/kg bw/day were selected for middle and low dose levels, respectively, using a common ratio of 2.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice daily (before and after each administration) during the administration period and once daily during the rest of the experimental period.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed on Days 0, 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 of gestation.
Body weight gain during Day 0 to 5, 5 to 20 and 0 to 20 of gestation were calculated.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The amount of food including the feeder was measured on Days 0, 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 of gestation. However, only the supplied food was measured on Day 0 of gestation, and only the remaining food on Day 20 of gestation. The mean daily food consumption was calculated using the weights of supplied and remained diets.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 (by exsanguination from the abdominal aorta under isoflurane anesthesia)
- Gross macroscopy was performed on all surviving animals: the external surfaces and orifices were observed, and the organs and tissues in the abdominal, thoracic, pelvic and cranial cavities were examined macroscopically.
- The thyroid glands of all survival dams were collected and weighed. On the basis of the body weight on the day of necropsy, relative weights were calculated.
- Histopathology was performed on thyroid glands.

HORMONE ANALYSES: Yes
- Blood was collected from each animal at necropsy. TSH, T3 and T4 levels were determined by ELISA.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
The gravid uterus (including the uterine contents) was weighed. On the basis of the body weight on the day of necropsy, their relative weights were calculated. In addition, the ovaries and uterine contents were examined, and the number of corpora lutea graviditatis, number of implantations, number of live fetuses and number of resorbed and dead fetuses (number of early dead embryos: deciduoma, number of late dead embryos: morphologically indistinct dead embryos with placenta and amnion, number of dead fetuses: including macerated fetuses), based on the implantation rate, live fetus rate and resorbed and dead fetus rate were calculated. The placentas of dams with live fetuses were weighed after gross examination for anomalies. The mean placental weight for each sex per litter was calculated. The uterus of the dam which was not pregnant (control animal) was stained with 10 vol% ammonium sulfide solution to confirm absence of implantation sites.

Fetal examinations:

The anogenital distance (AGD) of each fetus was measured using digital caliper. The mean AGD per sex per litter was calculated. All live fetuses were examined externally, and the incidence of external abnormalities per litter in each litter was calculated. The sex of each fetus was determined, and the sex ratio in each litter was calculated. Each fetus was weighed individually, and the mean fetal weight per sex per litter was calculated.
Approximately half of the live fetuses of a litter were examined for internal organ defects. Their head and abdomen were microscopically examined according to the modified Wilson method and their thorax was examined according to Nishimura’s microscopic necropsy method.
The remaining half of the live fetuses of a litter were stained for skeletal anomalies according to the modified Dawson method (Alizarin red S stain). Abnormalities were classified as skeletal malformations and variations, and the degree of ossification (number of ossified bones in the cervical centrum, number of incompletely ossified and unossified sternebra, number of ossified bones in metacarpal, fore proximal phalanx, middle phalanx, distal phalanx, metatarsal, hind proximal phalanx, middle
phalanx and distal phalanx, number of ossified bones in sacrococcygeal vertebra and other regions with incomplete ossification or unossification).

Statistics:

The body weight, body weight gain, food consumption, organ weights (absolute and relative), hormones, number of corpora lutea graviditatis, number of implantations, number of live fetuses, number of resorbed and dead fetuses (number of implantation sites), AGD, fetal weight, placental weight and degree of ossification were analyzed by Bartlett’s test for equality of variance. When the Bartlett’s test showed homoscedasticity (not significantly different), the data were analyzed by Dunnett’s multiple comparison test to assess the statistical significance of difference between the control group and each test substance-treated group. When the Bartlett’s test showed heteroscedasticity (significantly different), the data were analyzed by the Steel’s test to assess the statistical significance of difference between the control group and each test substance-treated group.
The implantation rate, live fetus rate, resorbed and dead fetus rate, sex ratio, and incidences of external anomalies, visceral abnormalities, skeletal variations, skeletal malformations and incomplete ossification were analyzed by Steel’s test to assess the statistical significance of difference between the control group and each test substance-treated group.
The significance level of the Bartlett's test was two-sided 5%. The significance levels of the other tests were two-sided 5% and 1%.
No statistical tests were used for clinical signs and gross and histopathological findings.

Indices:

Implantation rate: (Number of implantations/number of corpora lutea graviditatis) × 100
Live fetus rate: (Number of live fetuses/number of implantations) × 100
Resorbed and dead fetus rate: (Number of dead embryos and fetuses/number of implantations) × 100
Incidence of external abnormalities: (Number of abnormal fetuses / number of fetuses observed) × 100
Sex ratio: (Surviving male fetuses / surviving male and female fetuses) × 100
Incidence of visceral abnormalities: (Number of abnormal fetuses / number of fetuses observed) × 100
Incidence of skeletal malformation: (Number of fetuses with malformation / number of fetuses observed) × 100
Incidence of skeletal variation: (Number of fetuses with variation / number of fetuses observed) × 100
Incidence of incomplete ossification: (Number of abnormal fetuses/ number of fetuses observed) × 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Irregular respiration and abnormal respiratory noise were observed sporadically in two dams in the 400 mg/kg bw/day group during the latter half of the gestation period.

Mortality:

no mortality observed

Description (incidence):

No animals died in any group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 400 mg/kg bw/day group, the mean body weight on Day 20 of gestation was statistically significantly lower compared to the control group (-5%, p=< 0.05). The body weight gains from Day 5 to 20 of gestation and from Day 0 to 20 of gestation were significantly lower than those in the control group (-16% and -14%, respectively, both p=< 0.01). Moreover, in the 400 mg/kg bw/day group, the mean body weights on Days 6 and 7 were decreased from Day 5 of gestation (start day of administration), and a lower tendency of mean body weight was observed from Day 8 to 19 of gestation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the 200 and 400 mg/kg bw/day groups, the mean daily food consumption was significantly lower than that in the control group on Day 6 of gestation (Day 5 to 6 of gestation). In addition, in the 400 mg/kg bw/day group, significantly lower daily food consumption was observed from Day 7 to 12 and on Day 18 of gestation.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the levels of T4, T3 and TSH between each treatment group and the control group. The results are summarized in a tabel below.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the absolute and relative weights of thyroid glands and gravid uterus between treatment groups and control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the observation of placentas, enlarged placenta, white patch in the placenta and placenta accreta were observed in the control, 100 and 400 mg/kg bw/day groups, respectively. However, these findings were not considered to be treatment-related because each finding was observed in only one dam.

White patches on the kidneys were observed in 1/23 and 21/23 dams in the 200 and 400 mg/kg bw/day groups, respectively. Furthermore, in the 400 mg/kg bw/day group, atrophic thymus and wasting of whole body was observed in one dam. All other findings, brown patches on the lungs, hepatodiaphragmatic nodule of the liver and focal depression of the kidneys were observed sporadically in all groups. These findings were often observed in the rats of this strain, therefore, these were judged to be spontaneous occurring lesions.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No abnormal findings were noted in the thyroid glands in any group.

Number of abortions:

no effects observed

Description (incidence and severity):

The Caesarean section data are summarized in a tabel below.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

One female in the control group was found to be non-pregnant.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The Caesarean section data are summarized in a tabel below. There were no differences in average body weight of male and female pups between the control and exposed groups.

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The percentage males was 47.6% in the control group and 48.8%, 49.2% and 54.4% in the low, mid and high dose groups, respectively.

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External abnormalities were noted in 0 (0.0%), 2 (0.6%), 0 (0.0%) and 0 (0.0%) fetuses in the control, 100, 200 and 400 mg/kg bw/day groups, respectively. There were no statistically significant differences in the incidences of fetuses with external abnormalities between each treatment group and control group. The findings, gastroschisis and acaudate, were observed in one fetus each at 100 mg/kg bw/day. However, these findings were not considered to be test substance treatment-related, because each finding was observed in only one fetus and not observed in the 200 and 400 mg/kg bw/day groups.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The results of the skeletal examinations are summarized in tabular form and included as attachment.
Skeletal malformations were noted in 0 (0.0%), 1 (0.5%), 0 (0.0%) and 2 (1.2%) fetuses in the control, 100, 200 and 400 mg/kg bw/day groups, respectively. There were no statistically significant differences in the incidences of fetuses with skeletal malformations between each treatment group and control group. As the findings, sternoschisis was observed in the 100 mg/kg bw/day group, short rib and fused sternebra were observed in each one fetus of the 400 mg/kg bw/day groups.
Skeletal variations were noted in 8 (5.0%), 9 (4.9%), 13 (7.1%) and 8 (4.7%) fetuses in the control, 100, 200 and 400 mg/kg bw/day groups, respectively. There were no statistically significant differences in the incidences of fetuses with skeletal variations between each treatment group and control group. Findings included short supernumerary rib, wavy rib and lumbarization of sacral vertebra in the control or treatment groups, but there were no statistical significances in the incidences of these findings.
Retarded ossification was noted in 86 (57.3%), 89 (53.2%), 100 (53.9%) and 102 (57.1%) fetuses in the control, 100, 200 and 400 mg/kg bw/day groups, respectively. There were no statistically significant differences in the number of fetuses with retarded ossification or in the incidences of findings between each treatment group and control group. Findings included incomplete ossification of the frontal, incomplete ossification of the hyoid, unossified hyoid, incomplete ossification of the interparietal, incomplete ossification of the parietal, bipartite ossification of the sternebra, incomplete ossification of the cervical arch, bipartite ossification of the thoracic centrum, dumbbell ossification of the thoracic centrum, incomplete ossification of the thoracic centrum, unossified thoracic centrum, incomplete ossification of the lumbar arch, bipartite ossification of lumbar centrum, incomplete ossification of the lumbar centrum, incomplete ossification of the sacral arch, unossified sacral centrum, incomplete ossification of the ischium, incomplete ossification of pubis and unossified pubis.
There were no statistically significant differences in number of unossified or incomplete ossification of sternebrae, numbers of ossified metacarpal, metatarsal, fore/hind proximal, middle and distal phalanx, cervical vertebral centrum and sacrococcygeal vertebra between each treatment group and control group.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The findings are summarized in tabular form and included as attachment.
Visceral abnormalities were noted in 14 (9.6%), 13 (7.8%), 14 (7.4%) and 18 (10.6%) fetuses in the control, 100, 200 and 400 mg/kg bw/day groups, respectively. There were no statistically significant differences in the incidences of fetuses with visceral abnormalities between each treatment group and control group. Findings included thymic remnant in the neck, persistent left umbilical artery, elongate innominate, malpositioned subclavian branch, retroesophageal subclavian, misshapen liver, dilated renal pelvis, small kidney and misshapen plate rugae in the control or treatment groups, but there were no statistical significance in the incidences of these findings.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

Development

Effect level:

>= 400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed on development up to and including the highest dose tested (400 mg/kg bw/day)

Remarks on result:

other: Testing was done up to and including maternal toxic levels

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Tabel: Hormone measurements on GD 20

Dose level (mg/kg bw/day)	T4 (ng/mL)	T3 (ng/mL)	TSH (ng/mL)
0	13.61 ± 5.00 [22]	0.397 ± 0.132N [20]	1.99 ± 0.47N [16]
100	13.08 ± 4.82 [23]	0.416 ± 0.219 [18]	2.91 ± 2.75 [15]
200	12.34 ± 6.83 [23]	0.340 ± 0.097 [20]	2.27 ± 0.97 [18]
400	12.15 ± 6.52 [22]	0.366 ± 0.157 [20]	2.82 ± 1.65 [20]

Mean ± S.D.

No significant difference from the control.

N: Non parametric analysis

Values in [ ] parentheses expresses number of animals used for the calculation in each parameters

 

Tabel: Caesarean section data

		Controls	100 mg/kg bw/day	200 mg/kg bw/day	400 mg/kg bw/day
No. of animals examined	Total	22	23	23	23
No. of dams with live fetuses	Total	22	23	23	23
No. of corpora lutea	Total (mean±SD)	358 (16.3±1.8)	359 (15.6±2.1)	376 (16.3±1.9)	381 (16.6±3.1)
No. of implantations	Total (mean ± SD)	335 (15.2±2.9)	343 (14.9±2.1)	372 (16.2±1.7)	363 (15.8±2.1)
No. of dead fetuses	Total (mean ± SD)	27 (1.2±1.3)	17 (0.7±0.9)	14 (0.6±0.9)	15 (0.7±0.8)
Early resorptions	Total (mean ± SD)	27 (1.2±1.3)	15 (0.7±0.7)	13 (0.6±0.9)	14 (0.6±0.7)
Late resorptions	Total (mean ± SD)	0	1 (0.0±0.2)	1 (0.0±0.2)	1 (0.0±0.2)
Dead fetuses	Total (mean ± SD)	0	1 (0.0±0.2)	0	0
No. of live fetuses	Total (mean ± SD)	308 (14.0±3.2)	326 (14.2±2.1)	358 (15.6±1.9)	348 15.1±2.0)
Sex ratio	Male:female (% males)	147:161 (47.6±11.2)	159:167 (48.8±15.5)	177:181 (49.2±13.4)	188:160 (54.4±11.7)
Body weight (g)	Males (mean ± SD)	3.79±0.30	3.80±0.29	3.82±0.26	3.61±0.41
Body weight (g)	Females (mean ± SD)	3.57±0.26	3.57±0.30	3.63±0.16	3.43±0.40
Placental weight (g)	Male (mean ± SD)	0.49±0.13	0.44±0.04	0.46±0.04	0.43±0.05#
Placental weight (g)	Female (mean ± SD)	0.46±0.12	0.42±0.03	0.43±0.03	0.41±0.05
Anogenital distance (mm)	Male (mean ± SD)	2.9±0.2	2.9±0.2	2.9±0.3	2.8±0.3
Anogenital distance (mm)	Female (mean ± SD)	1.2±0.2	1.2±0.2	1.2±0.2	1.2±0.2
No. of live fetuses with external anomalies	Total (%)	0 (0)	2 (0.6)	0 (0)	0 (0)

Significant difference from the control; # P ≤ 0.05 (Steel’s test)

Conclusions:

Based on the results of a prenatal toxicity study, performed according to OECD guideline 414 and GLP principles, the maternal NOAEL was 100 mg/kg bw/day and the developmental NOAEL was at least 400 mg/kg bw/day, the highest dose tested.

Executive summary:

A prenatal toxicity study with exposure of rats via oral gavage was performed with XDI according to OECD guideline 414 and GLP principles. Based on the results of a dose range finder study, the dose levels were set at 100, 200 and 400 mg/kg bw/day. Corn oil was used as a vehicle, dose formulation analyses were included to verify correct dosing of the animals. The rats were dosed from GD 5 to 19, and sacrificed on GD20.

No mortality occurred during the study. Maternal toxicity was found in the mid and the high dose groups, and consisted of lower food consumption (mid dose: GD 5 -6, high dose group GD 5 to the end of the study). For the high dose group, a statistically significant decrease in body weight gain was found. No treatment related clinical signs were observed. At macroscopy white patches were found on the kidneys of animals dosed at 200 and 400 mg/kg bw/day. No effects were found in thyroids (weights and histopathology), or in the levels of thyroid hormones (T3, T4 and TSH). There were no test substance treatment-related effects on the sex ratio, body weights of live fetuses, anogenital distance (AGD), findings of external, visceral and skeletal examinations.

In conclusion, the maternal NOAEL was found to be 100 mg/kg bw/day and the developmental NOAEL was at least 400 mg/kg bw/day, the highest dose tested.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

400 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Two reliable studies are available (both studies have Klimisch score 1).

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In an OECD 422 study, rats were exposed to XDI orally to 0, 12.5, 50 and 200 mg/kg bw/day. 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 parental NOAEL is 50 mg/kg bw/d and the developmental/fertility NOAEL is >= 200 mg/kg bw/d.

A prenatal toxicity study with exposure of rats via oral gavage was performed with XDI according to OECD guideline 414 and GLP principles. Based on the results of a dose range finder study, the dose levels were set at 100, 200 and 400 mg/kg bw/day. Corn oil was used as a vehicle, dose formulation analyses were included to verify correct dosing of the animals. The rats were dosed from GD 5 to 19, and sacrificed on GD20. No mortality occurred during the study. Maternal toxicity was found in the mid and the high dose groups, and consisted of lower food consumption (mid dose: GD 5 -6, high dose group GD 5 to the end of the study). For the high dose group, a statistically significant decrease in body weight gain was found. No treatment related clinical signs were observed. At macroscopy white patches were found on the kidneys of animals dosed at 200 and 400 mg/kg bw/day. No effects were found in thyroids (weights and histopathology), or in the levels of thyroid hormones (T3, T4 and TSH). There were no test substance treatment-related effects on the sex ratio, body weights of live fetuses, anogenital distance (AGD), findings of external, visceral and skeletal examinations.

In conclusion, the maternal NOAEL was found to be 100 mg/kg bw/day and the developmental NOAEL was at least 400 mg/kg bw/day, the highest dose tested.

Justification for classification or non-classification

As the current data does not indicate any effect of XDI on reproduction and development, XDI is not classified according to the CLP Regulation (EC) No. 1272/2008 for reproduction and developmental toxicity.