4-[(2,5-dichlorophenyl)azo]-3-hydroxy-N-phenylnaphthalene-2-carboxamide

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

PR022

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test with the test item administered by oral gavage in Sprague Dawley rats (12/sex/dose) was performed according to OECD TG 422. The dose levels for this study were 0, 100, 300 and 1000 mg/kg/day for a total of 37 days (males) and through mating, gestation and delivery until day 4 of lactation (females).

There were no effects on reproduction or development; therefore, the NOAEL for the reproductive/developmental toxicity was considered to be 1000 mg/kg/day in both parental animals and offspring.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (according to OECD 422 and GLP)

Justification for type of information:

See rationale and justification for the analogue read-across approach for the registration of the nanoform of Pigment Red 002 (chapter 13).

Reason / purpose for cross-reference:

read-across source

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:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc. (Atsugi Breeding center)
- Age at study initiation: 8 weeks
- Weight at study initiation: males 309-355 g, females: 206 to 232 g
- Housing: individually; mating period: 1 male, 1 female; 1 litter during lactation
- Diet: autoclaved pellet die for laboratory animals (CRF-1, Oriental Yeast Industry Co., LTD), ad libitum
- Water: sterile tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-2
- Humidity (%): 55 +/- 15
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % sodium-carboxymethylcellulose (CMC-NA) aqueous solution containing 0.1 % Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of test substance : once or twice a week
- Storage temperature of suspension: 4°C

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Amount of vehicle (if gavage): 10 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At the first preparation, the dosing formulations were analyzed by HPLC and it was confirmed that the concentrations were within the target value +/-10%.

Duration of treatment / exposure:

males: from 14 days before mating and through the mating period until the day before necroscopy for a total of 37 days
females: from 14 days beformating, through the matingand gestation periods and delivery until day 4 of lactation for a total of 42 to 47 days. Non-delivering females, however, were treated until the day before necropsy.

Frequency of treatment:

daily

Details on study schedule:

no data

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: highest dose is the upper limit dose level stipulated in the OECD guideline
- Rationale for animal assignment (if not random): stratified-by-weight randomization methodon the basis of the body weights to get almost the same mean body weights for each group.

Positive control:

none

Parental animals: Observations and examinations:

For deatails on parental animals see section 7.5.1 (repeated dose toxicity oral).

Oestrous cyclicity (parental animals):

From the first day of dosing to the start of mating, the estrous cycle was examined by collecting vaginal smears every day in the morning for all females of each group, and the mean estrous cycle length of each female was calculated. Females that had an estrous cycle length other than 4 to 6 days were judged as having irregular estrous cycles.

Sperm parameters (parental animals):

no data

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS: yes

Postmortem examinations (parental animals):

For deatials on parental animals see section 7.5.1 (repeated dose toxicity oral).

Examination of the ovary and uteri of the dams for the numbers of corpora lutea and implantations.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic and microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Statistics:

Multiple comparison test:
Body weight, body weight gain, food consumption, hematology, blood chemistry, organ weight, number of corpora lutea, number of implantations, number of offspring

Kruskal-Wallis test and Dunnett-type multiple comparison test:
Days until copulation, number of estrus stages without copulation, mean estrous cycle length, gestation length, implantation index, delivery index, live birth index, incidence of offspring with external anomaly, viability index an Day 4

Chi-square test: Histopathological findings

Fisher's exact probability test:
Incidence of females with irregular estrous cycles, copulation index, fertility index, gestation index, sex ratio (rnale/fernale), incidence of dams with externally abnormal offspring

Reproductive indices:

(1) Days until copulation: Number of days from the start of mating to detection of copulation
(2) Number of estrus stages without copulation
(3) Copulation index (%): (Number of animals with successful copulation/Number of animals paired) x 100
(4) Fertility index (%): (Number of pregnant animals/Number of animals with successful copulation) x 100
(5) Gestation length: Days until completion of delivery from Day 0 of gestation
(6) Gestation index (%): (Number of females with live offspring/Number of pregnant females) x 100
(7) Implantation index (%): (Number of implantations/Number of corpora lutea) x 100
(8) Delivery index (%): (Total number of offspring born/Number of implantations) x 100

Offspring viability indices:

(1) Live birth index (%): (Number of offspring born alive/ Total number of offspring born) x 100
(2) Viability index on Day 4 (%): (Number of live offspring on Day 4/ Number of offspring born alive) x 100

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
In the estrous cycle examination, there was no significant difference in the mean estrous cycle length between the control and treated groups. Moreover, there were no females with irregular estrous cycles in any group including the control group.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
As a results of mating, all the mating pairs of each group copulated including the control group, and there was no significant difference in the copulation index, nor were there any in the number of estrus stages without copulation or the days until copulation between the control and treated groups. Moreover, there were only 1 and 2 non-pregnant females in the 100 and 300 mg/kg groups, respectively, and no significant difference was found in the fertility index between the control and treated groups.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no effects on reproduction or development up to the highest dose tested.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

1) Observation of offspring
There were no significant differences in the number of offspring born or born alive, sex ratio, live birth index or viability index on Day 4 between the control and each treated group. In the clinical observation, subcutaneous hemorrhage and abdominal hemorrhage were observed in 1 or 2 pups each in the control and 100 mg/kg groups, respectively; however, they were judged not treatment-related because of the lack of dose-dependency in their incidences. Although loss of suckling was observed in some neonates on the birthday in the control, 300 and 1000 mg/kg groups, it was judged not treatment-related because all pups were confirmed to have been suckled by the dams on the next day. Moreover, no abnormal external features were found in neonates in any group including the control group.

2) Body weight
There were no significant differences in body weight or body weight gain of either sex between the control and treated groups.

3) Necropsy findings
Hemorrhage in the abdominal cavity, and white and yellow patches on the liver were observed only in 1 male in the 100 mg/kg group, as was dilatation of the renal pelvis in 2 males in the 1000 mg/kg group. However, they were judged not treatrnent-related, because the same findings did not occur frequently in the 1000 mg/kg group. Moreover, there were no abnormal findings in pups that died until postnatal Day 4.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no effects on reproduction or development up to the highest dose tested.

Reproductive effects observed:

not specified

Conclusions:

Under the conditions of the test, there were no effects on reproduction or development; therefore, the NOAEL for the reproductive / developmental toxicity was considered to be 1000 mg/kg bw/day in both parental animals and offspring.

Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test with the test item administered by oral gavage in Sprague Dawley rats (12/sex/dose) was performed according to OECD TG 422. The dose levels for this study were 0, 100, 300 and 1000 mg/kg/day for a total of 37 days (males) and through mating, gestation and delivery until day 4 of lactation (females).

There were no effects on reproduction or development; therefore, the NOAEL for the reproductive/developmental toxicity was considered to be 1000 mg/kg/day in both parental animals and offspring.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (according to OECD 422 and GLP)

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:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc. (Atsugi Breeding center)
- Age at study initiation: 8 weeks
- Weight at study initiation: males 309-355 g, females: 206 to 232 g
- Housing: individually; mating period: 1 male, 1 female; 1 litter during lactation
- Diet: autoclaved pellet die for laboratory animals (CRF-1, Oriental Yeast Industry Co., LTD), ad libitum
- Water: sterile tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-2
- Humidity (%): 55 +/- 15
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % sodium-carboxymethylcellulose (CMC-NA) aqueous solution containing 0.1 % Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of test substance : once or twice a week
- Storage temperature of suspension: 4°C

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Amount of vehicle (if gavage): 10 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At the first preparation, the dosing formulations were analyzed by HPLC and it was confirmed that the concentrations were within the target value +/-10%.

Duration of treatment / exposure:

males: from 14 days before mating and through the mating period until the day before necroscopy for a total of 37 days
females: from 14 days beformating, through the matingand gestation periods and delivery until day 4 of lactation for a total of 42 to 47 days. Non-delivering females, however, were treated until the day before necropsy.

Frequency of treatment:

daily

Details on study schedule:

no data

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: highest dose is the upper limit dose level stipulated in the OECD guideline
- Rationale for animal assignment (if not random): stratified-by-weight randomization methodon the basis of the body weights to get almost the same mean body weights for each group.

Positive control:

none

Parental animals: Observations and examinations:

For deatails on parental animals see section 7.5.1 (repeated dose toxicity oral).

Oestrous cyclicity (parental animals):

From the first day of dosing to the start of mating, the estrous cycle was examined by collecting vaginal smears every day in the morning for all females of each group, and the mean estrous cycle length of each female was calculated. Females that had an estrous cycle length other than 4 to 6 days were judged as having irregular estrous cycles.

Sperm parameters (parental animals):

no data

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS: yes

Postmortem examinations (parental animals):

For deatials on parental animals see section 7.5.1 (repeated dose toxicity oral).

Examination of the ovary and uteri of the dams for the numbers of corpora lutea and implantations.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic and microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Statistics:

Multiple comparison test:
Body weight, body weight gain, food consumption, hematology, blood chemistry, organ weight, number of corpora lutea, number of implantations, number of offspring

Kruskal-Wallis test and Dunnett-type multiple comparison test:
Days until copulation, number of estrus stages without copulation, mean estrous cycle length, gestation length, implantation index, delivery index, live birth index, incidence of offspring with external anomaly, viability index an Day 4

Chi-square test: Histopathological findings

Fisher's exact probability test:
Incidence of females with irregular estrous cycles, copulation index, fertility index, gestation index, sex ratio (rnale/fernale), incidence of dams with externally abnormal offspring

Reproductive indices:

(1) Days until copulation: Number of days from the start of mating to detection of copulation
(2) Number of estrus stages without copulation
(3) Copulation index (%): (Number of animals with successful copulation/Number of animals paired) x 100
(4) Fertility index (%): (Number of pregnant animals/Number of animals with successful copulation) x 100
(5) Gestation length: Days until completion of delivery from Day 0 of gestation
(6) Gestation index (%): (Number of females with live offspring/Number of pregnant females) x 100
(7) Implantation index (%): (Number of implantations/Number of corpora lutea) x 100
(8) Delivery index (%): (Total number of offspring born/Number of implantations) x 100

Offspring viability indices:

(1) Live birth index (%): (Number of offspring born alive/ Total number of offspring born) x 100
(2) Viability index on Day 4 (%): (Number of live offspring on Day 4/ Number of offspring born alive) x 100

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
In the estrous cycle examination, there was no significant difference in the mean estrous cycle length between the control and treated groups. Moreover, there were no females with irregular estrous cycles in any group including the control group.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
As a results of mating, all the mating pairs of each group copulated including the control group, and there was no significant difference in the copulation index, nor were there any in the number of estrus stages without copulation or the days until copulation between the control and treated groups. Moreover, there were only 1 and 2 non-pregnant females in the 100 and 300 mg/kg groups, respectively, and no significant difference was found in the fertility index between the control and treated groups.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no effects on reproduction or development up to the highest dose tested.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

1) Observation of offspring
There were no significant differences in the number of offspring born or born alive, sex ratio, live birth index or viability index on Day 4 between the control and each treated group. In the clinical observation, subcutaneous hemorrhage and abdominal hemorrhage were observed in 1 or 2 pups each in the control and 100 mg/kg groups, respectively; however, they were judged not treatment-related because of the lack of dose-dependency in their incidences. Although loss of suckling was observed in some neonates on the birthday in the control, 300 and 1000 mg/kg groups, it was judged not treatment-related because all pups were confirmed to have been suckled by the dams on the next day. Moreover, no abnormal external features were found in neonates in any group including the control group.

2) Body weight
There were no significant differences in body weight or body weight gain of either sex between the control and treated groups.

3) Necropsy findings
Hemorrhage in the abdominal cavity, and white and yellow patches on the liver were observed only in 1 male in the 100 mg/kg group, as was dilatation of the renal pelvis in 2 males in the 1000 mg/kg group. However, they were judged not treatrnent-related, because the same findings did not occur frequently in the 1000 mg/kg group. Moreover, there were no abnormal findings in pups that died until postnatal Day 4.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no effects on reproduction or development up to the highest dose tested.

Reproductive effects observed:

not specified

Conclusions:

Under the conditions of the test, there were no effects on reproduction or development; therefore, the NOAEL for the reproductive / developmental toxicity was considered to be 1000 mg/kg bw/day in both parental animals and offspring.

Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test with the test item administered by oral gavage in Sprague Dawley rats (12/sex/dose) was performed according to OECD TG 422. The dose levels for this study were 0, 100, 300 and 1000 mg/kg/day for a total of 37 days (males) and through mating, gestation and delivery until day 4 of lactation (females).

There were no effects on reproduction or development; therefore, the NOAEL for the reproductive/developmental toxicity was considered to be 1000 mg/kg/day in both parental animals and offspring.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

PR022

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test with the test item administered by oral gavage in Sprague Dawley rats (12/sex/dose) was performed according to OECD TG 422. The dose levels for this study were 0, 100, 300 and 1000 mg/kg/day for a total of 37 days (males) and through mating, gestation and delivery until day 4 of lactation (females).

There were no effects on reproduction or development; therefore, the NOAEL for the reproductive/developmental toxicity was considered to be 1000 mg/kg/day in both parental animals and offspring.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (according to OECD 422 and GLP)

Justification for type of information:

See rationale and justification for the analogue read-across approach for the registration of the nanoform of Pigment Red 002 (chapter 13).

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: 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:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc. (Atsugi Breeding center)
- Age at study initiation: 8 weeks
- Weight at study initiation: males 309-355 g, females: 206 to 232 g
- Housing: individually; mating period: 1 male, 1 female; 1 litter during lactation
- Diet: autoclaved pellet die for laboratory animals (CRF-1, Oriental Yeast Industry Co., LTD), ad libitum
- Water: sterile tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-2
- Humidity (%): 55 +/- 15
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % sodium-carboxymethylcellulose (CMC-NA) aqueous solution containing 0.1 % Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of test substance : once or twice a week
- Storage temperature of suspension: 4°C

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At the first preparation, the dosing formulations were analyzed by HPLC and it was confirmed that the concentrations were within the target value +/-10%.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no data

Duration of treatment / exposure:

males: from 14 days before mating and throught he mating period until the day before necroscopy for a total of 37 days
females: from 14 days beformating, through the matingand gestation periods and delivery until day 4 of lactation for a total of 42 to 47 days. Non-delivering females, however, were treated until the day before necropsy.

Frequency of treatment:

daily

Duration of test:

till day 4 postnatally

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: highest dose is the upper limit dose level stipulated in the OECD guideline
- Rationale for animal assignment (if not random): stratified-by-weight randomization methodon the basis of the body weights to get almost the same mean body weights for each group.

Maternal examinations:

For details on parental animals see section 7.5.1 (repeated dose toxicity oral).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes microscopically

Fetal examinations:

- External examinations: Yes: (all)
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

Statistics:

Multiple comparison test:
Body weight, body weight gain, food consumption, hematology, blood chemistry, organ weight, number of corpora lutea, number of implantations, number of offspring

Kruskal-Wallis test and Dunnett-type multiple comparison test:
Days until copulation, number of estrus stages without copulation, mean estrous cycle length, gestation length, implantation index, delivery index, live birth index, incidence of offspring with external anomaly, viability index an Day 4

Chi-square test: Histopathological findings

Fisher's exact probability test:
Incidence of females with irregular estrous cycles, copulation index, fertility index, gestation index, sex ratio (rnale/fernale), incidence of dams with externally abnormal offspring

Indices:

Reproductive indices
(1) Days until copulation: Number of days from the start of mating to detection of copulation
(2) Number of estrus stages without copulation
(3) Copulation index (%): (Number of animals with successful copulation/Number of animals paired) x 100
(4) Fertility index (%): (Number of pregnant animals/Number of animals with successful copulation) x 100
(5) Gestation length: Days until completion of delivery from Day 0 of gestation
(6) Gestation index (%): (Number of females with live offspring/Number of pregnant females) x 100
(7) Implantation index (%): (Number of implantations/Number of corpora lutea) x 100
(8) Delivery index (%): (Total number of offspring born/Number of implantations) x 100
Offspring viability indices
(1) Live birth index (%): (Number of offspring born alive/ Total number of offspring born) x 100
(2) Viability index on Day 4 (%): (Number of live offspring on Day 4/ Number of offspring born alive) x 100

Historical control data:

no data

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: details see below

Details on maternal toxic effects:
Effects observed in females were judged not to be adverse, for details see section 7.5.1 (NOAEL 1000 mg/kg bw/d)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
1) Observation of offspring
There were no significant differences in the number of offspring born or born alive, sex ratio, live birth index or viability index on Day 4 between the control and each treated group. In the clinical observation, subcutaneous hemorrhage and abdominal hemorrhage were observed in 1 or 2 pups each in the control and 100 mg/kg groups, respectively; however, they were judged not treatment-related because of the lack of dose-dependency in their incidences. Although loss of suckling was observed in some neonates on the birthday in the control, 300 and 1000 mg/kg groups, it was judged not treatment-related because all pups were confirmed to have been suckled by the dams on the next day. Moreover, no abnormal external features were found in neonates in any group including the control group.

2) Body weight
There were no significant differences in body weight or body weight gain of either sex between the control and treated groups.

3) Necropsy findings
Hemorrhage in the abdominal cavity, and white and yellow patches on the liver were observed only in 1 male in the 100 mg/kg group, as was dilatation of the renal pelvis in 2 males in the 1000 mg/kg group. However, they were judged not treatrnent-related, because the same findings did not occur frequently in the 1000 mg/kg group. Moreover, there were no abnormal findings in pups that died until postnatal Day 4.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Development was not impaired at any dose level.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Under the conditions of the test there were no effects on reproduction or development; therefore, the NOAEL for the reproductive / developmental toxicity was considered to be 1000 mg/kg bw/day in both parental animals and offspring.

Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test with the test item administered by oral gavage in Sprague Dawley rats (12/sex/dose) was performed according to OECD TG 422. The dose levels for this study were 0, 100, 300 and 1000 mg/kg/day for a total of 37 days (males) and through mating, gestation and delivery until day 4 of lactation (females).

There were no effects on reproduction or development; therefore, the NOAEL for the reproductive/developmental toxicity was considered to be 1000 mg/kg/day in both parental animals and offspring.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (according to OECD 422 and GLP)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: 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:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc. (Atsugi Breeding center)
- Age at study initiation: 8 weeks
- Weight at study initiation: males 309-355 g, females: 206 to 232 g
- Housing: individually; mating period: 1 male, 1 female; 1 litter during lactation
- Diet: autoclaved pellet die for laboratory animals (CRF-1, Oriental Yeast Industry Co., LTD), ad libitum
- Water: sterile tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-2
- Humidity (%): 55 +/- 15
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % sodium-carboxymethylcellulose (CMC-NA) aqueous solution containing 0.1 % Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of test substance : once or twice a week
- Storage temperature of suspension: 4°C

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At the first preparation, the dosing formulations were analyzed by HPLC and it was confirmed that the concentrations were within the target value +/-10%.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no data

Duration of treatment / exposure:

males: from 14 days before mating and throught he mating period until the day before necroscopy for a total of 37 days
females: from 14 days beformating, through the matingand gestation periods and delivery until day 4 of lactation for a total of 42 to 47 days. Non-delivering females, however, were treated until the day before necropsy.

Frequency of treatment:

daily

Duration of test:

till day 4 postnatally

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: highest dose is the upper limit dose level stipulated in the OECD guideline
- Rationale for animal assignment (if not random): stratified-by-weight randomization methodon the basis of the body weights to get almost the same mean body weights for each group.

Maternal examinations:

For details on parental animals see section 7.5.1 (repeated dose toxicity oral).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes microscopically

Fetal examinations:

- External examinations: Yes: (all)
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

Statistics:

Multiple comparison test:
Body weight, body weight gain, food consumption, hematology, blood chemistry, organ weight, number of corpora lutea, number of implantations, number of offspring

Kruskal-Wallis test and Dunnett-type multiple comparison test:
Days until copulation, number of estrus stages without copulation, mean estrous cycle length, gestation length, implantation index, delivery index, live birth index, incidence of offspring with external anomaly, viability index an Day 4

Chi-square test: Histopathological findings

Fisher's exact probability test:
Incidence of females with irregular estrous cycles, copulation index, fertility index, gestation index, sex ratio (rnale/fernale), incidence of dams with externally abnormal offspring

Indices:

Reproductive indices
(1) Days until copulation: Number of days from the start of mating to detection of copulation
(2) Number of estrus stages without copulation
(3) Copulation index (%): (Number of animals with successful copulation/Number of animals paired) x 100
(4) Fertility index (%): (Number of pregnant animals/Number of animals with successful copulation) x 100
(5) Gestation length: Days until completion of delivery from Day 0 of gestation
(6) Gestation index (%): (Number of females with live offspring/Number of pregnant females) x 100
(7) Implantation index (%): (Number of implantations/Number of corpora lutea) x 100
(8) Delivery index (%): (Total number of offspring born/Number of implantations) x 100
Offspring viability indices
(1) Live birth index (%): (Number of offspring born alive/ Total number of offspring born) x 100
(2) Viability index on Day 4 (%): (Number of live offspring on Day 4/ Number of offspring born alive) x 100

Historical control data:

no data

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: details see below

Details on maternal toxic effects:
Effects observed in females were judged not to be adverse, for details see section 7.5.1 (NOAEL 1000 mg/kg bw/d)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
1) Observation of offspring
There were no significant differences in the number of offspring born or born alive, sex ratio, live birth index or viability index on Day 4 between the control and each treated group. In the clinical observation, subcutaneous hemorrhage and abdominal hemorrhage were observed in 1 or 2 pups each in the control and 100 mg/kg groups, respectively; however, they were judged not treatment-related because of the lack of dose-dependency in their incidences. Although loss of suckling was observed in some neonates on the birthday in the control, 300 and 1000 mg/kg groups, it was judged not treatment-related because all pups were confirmed to have been suckled by the dams on the next day. Moreover, no abnormal external features were found in neonates in any group including the control group.

2) Body weight
There were no significant differences in body weight or body weight gain of either sex between the control and treated groups.

3) Necropsy findings
Hemorrhage in the abdominal cavity, and white and yellow patches on the liver were observed only in 1 male in the 100 mg/kg group, as was dilatation of the renal pelvis in 2 males in the 1000 mg/kg group. However, they were judged not treatrnent-related, because the same findings did not occur frequently in the 1000 mg/kg group. Moreover, there were no abnormal findings in pups that died until postnatal Day 4.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Development was not impaired at any dose level.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Under the conditions of the test there were no effects on reproduction or development; therefore, the NOAEL for the reproductive / developmental toxicity was considered to be 1000 mg/kg bw/day in both parental animals and offspring.

Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test with the test item administered by oral gavage in Sprague Dawley rats (12/sex/dose) was performed according to OECD TG 422. The dose levels for this study were 0, 100, 300 and 1000 mg/kg/day for a total of 37 days (males) and through mating, gestation and delivery until day 4 of lactation (females).

There were no effects on reproduction or development; therefore, the NOAEL for the reproductive/developmental toxicity was considered to be 1000 mg/kg/day in both parental animals and offspring.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

No Classification, as no adverse effects were observed.