1,4-bis(mesitylamino)anthraquinone

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

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:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed GLP compliant OECD guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (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 Italy S.p.A., Calco (Lecco), Italy
- Age at study initiation: 8-9 weeks
- Weight at study initiation: (P) Males: 317-322g; Females: 210-216 g;
- Housing:
From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulphone solid bottomed cages measuring approximately 59.5x38x20 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During the mating period, animals were housed on the basis of one male to one female in clear polycarbonate cages measuring approxiamtely 43x27x18 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily.
The males were re-caged after mating as they were before mating.
After mating, the females were transferred to individual clear polycarbonate solid bottomed cages measuring approxiamtely 43x27x18 cm
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20-24
- Humidity (%):40-70
- Air changes (per hr):15-20
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From: To:05 July 2012-05 September 2012

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The required amount of the test item was suspended in the vehicle (corn oil), brought to the final volume appropriate for each concentration (concentrations of 20, 60 and 200 mg/mL) and kept under magnetic stirrer at room temperature prior to use and until the time of dosing of the last animal.
Concentrations were calculated and expressed in terms of test item as supplied.

Details on mating procedure:

Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).
The female was paired with the same male until positive identification occurred.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable (content check and homogeneity) and the stability of formulation was satisfactory. Results of the analyses were within the limits of acceptance.
The stability was found to be 24 hours at room temperature in the concentration range of 20 to 200 mg/mL.
Samples of the formulations prepared on Day 1 and on Week 4 of the study were also analysed to check the concentration and homogeneity. Results of the analyses were within the limits of acceptance.
Chemical analyses were carried out by the Analytical Chemistry Department at RTC, using a validated spectrophotometric detection in the range from 1.0 to 250 mg/mL.
The software used for this activity was Empower Probuild No. 2154.

Duration of treatment / exposure:

Males were dosed for 32 days (including 2 weeks prior to pairing during the pre pairing period and approximately 2 weeks post pairing). Females were dosed 2 weeks before pairing, during the pairing period, gestation period and up to Day 3 post partum.

Frequency of treatment:

Males were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter through the day before necropsy.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum or the day before sacrifice. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.

Details on study schedule:

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 32 days.
Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum or until the day before necropsy.
The following investigations were performed on parental animals of all groups: body weight, clinical signs, food consumption, oestrous cycle, mating performance, litter data, macroscopic observations, organ weights and histopathological examination of abnormalities. Histopathological examination of testes, epididymides and ovaries was performed only on control and high dose groups.

Remarks:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Dose levels had been selected in consultation with the Sponsor based on information from a previous non GLP compliant dose range finding study.

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Daily

BODY WEIGHT: Yes
Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum.


FOOD CONSUMPTION: Yes
Food consumption was recorded at weekly intervals whenever possible, for each cage of rats from allocation to pairing. For female animals, food consumption was also recorded on Days 7, 14 and 20 post coitum, starting from Day 0 post coitum and on Day 4 post partum (starting from Day 1 post partum).

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily in the morning from the first day of treatment and up to the end of the mating period, until a positive identification of mating is made. The vaginal smear data were examined to determine the following:

a) anomalies of the oestrous cycle;
b) the pre-coital interval (i.e., the number of nights paired prior to the detection of mating).

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight
The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed in control and high dose males.

Litter observations:

A parturition check was performed from Day 20 post coitum. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring are first detected in the cage was considered Day 0 post partum.
As soon as possible, after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were identified.
Live pups were individually weighed on Days 1 and 4 post partum.
Pups dying during the lactation period were weighed before the despatch to necropsy.
Observations were performed once daily for all litters.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All animals after 32 days ofdosing
- Maternal animals: all live females with pups were killed on Day 4 post partum with pups; non rpegnant female after day 25 of post coitum

GROSS NECROPSY
The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.

Females:
All females were examined also for the following:

a) number of visible implantation sites;
b) number of corpora lutea (pregnant animals).

Uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.


Organ weights

From all animals completing the scheduled test period, the organs indicated below were dissected free of fat and weighed. The ratios of organ weight to terminal body weight were calculated for each animal.

Tissues fixed and preserved

Samples of all the tissues listed in section 4.4.6 were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in modified Davidson's fluid) from all animals.

Histopathological examination and staging of spermatogenic cycle

The tissues required for histopathological examination are listed below. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre
thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous
epithelium (staging of spermatogenic cycle) was performed in control and high dose males.

In the first instance the examination was restricted as detailed below:

a) Tissues specified in section 4.4.6 from all animals in the control and high dose group killed at term.
b) Tissues specified in section 4.4.6 from all animals dying during the treatment period.
c) All abnormalities in all groups.

Postmortem examinations (offspring):

All pups found dead in the cage were examined for external and internal abnormalities.
All live pups were killed on Day 4 post partum and examined for external abnormalities and sex confirmation by gonadal inspection.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p<0.05.

Reproductive indices:

Group mean values were calculated for all parameters. Data from females not pregnant were excluded from group mean calculations as considered appropriate by the Study Director.

The following reproductive indices were calculated:

Males

Copulatory Index (%) = (no. of animals mated x 100) / (no. of animals paired)

Fertility Index (%) = (no. of males which induced pregnancy x 100) / (no. of males paired)


Females

Copulatory Index (%) = (no. of animals mated x 100) / (no. of animals paired)

Fertility Index (%) = (no. of pregnant females x 100) / (no. of females paired)


Males and females

Copulatory Interval = Mean number of days between pairing and mating


Females

Pre-birth loss was calculated as a percentage from the formula:
(((No. of visible implantations) - (total litter size at birth)) x 100) / (No. of visible implantations)

Pup loss at birth was calculated as a percentage from the formula:
(((Total litter size) - (live litter size)) x 100) / (Total litter size)

Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula:
(((Total litter size at birth) - (live litter size at Day 4)) x 100) / (Total litter size at birth)

Sex ratios were calculated at birth and on Day 4 post partum and presented as the percentage of males per litter.

Offspring viability indices:

Pup loss at birth
Cumulative pup loss on Day 4 post partum

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:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Mortality and fate of females:

One control female and one low dose female were found dead. On the basis of the macroscopic and microscopic observations, the cause of deaths is attributable to a mis-dosing.
One low dose female was not pregnant and one mid-dose female showed unilateral implantation. The number of females with live pups on Day 4 post partum was 9 in the control group, 8 in the low dose group and 10 in each of the mid- and high dose groups.


Clinical signs:

As consequence of the colour of the test item blue staining of the bedding material and tail was noted in the animals of mid- and high dose groups.


Body weight and body weight gain:

No signs of toxicological significance were seen in body weight or body weight gain during the study in treated animals compared to controls.


Food consumption:

No effects on food consumption were seen.


Oestrus cycle, mating performance and reproductive parameters:

No treatment-related anomalies were noted in the oestrus cycle of treated females when compared to controls. Pre-coital interval, copulation plugs, copulatory and fertility index were similar between treated and control animals.


Implantation, pre-birth loss data and gestation length:

No differences were observed in treated and control groups for these parameters.


Litter data and sex ratio:

Litter data and sex ratios at birth and on Day 4 post partum were unaffected by treatment.


Clinical signs of pups:

Pre-weaning clinical signs did not show treatment-related effects.


Necropsy findings in pups:

Necropsy findings in decedent pups and pups sacrificed on Day 4 post partum did not reveal any treatment-related effect.


Terminal body weight and organ weights:

Terminal body weight and organ weights were unaffected by treatment in both sexes.


Macroscopic and microscopic observations including examination of spermatogenic cycle:

The lesions detected in some animals were also noted in untreated laboratory rats; they were therefore considered to be incidental and/or spontaneous in origin.
No treatment-related changes were seen in testis, epididymides (including spermatogenic cycle) and ovaries evaluated in high dose males or females receiving the test substance.


Conclusions:

On the basis of the above mentioned results, the dosage of 1000 mg/kg/day of the test item was established as the NOAEL (No Observed Adverse Effect Level) for animals of both sexes.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects 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

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects up to the highest dose tested

Reproductive effects observed:

not specified

Conclusions:

Clinical signs, body weight and food consumption were unaffected by treatment in both sexes.
Reproductive parameters such as fertility index, pre-coital interval and copulatory index did not show significant differences in treated groups compared to controls.
Gestation length, litter data, sex ratios and implantation losses (pre and post) were unaffected by treatment.
At macroscopic and microscopic examination, no treatment related lesions were seen.
On the basis of the results obtained in this OECD Reproduction / Developmental Toxicity Screening Test, the maximum dosage of 1000 mg/kg/day of the test item was established as the NOAEL (No Observed Adverse Effect Level) for parental animals of both sexes (general toxicity and reproduction) and for the offspring (developmental toxicitxy).

Executive summary:

Study design

The toxic effects on rats after repeated dosing, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition, development of the concepts and early lactation were investigated.

The dosage groups were as follows:

Group Number	Treatment (mg/kg/day)	Number of animals
1	0	10M+10F
2	100	10M+10F
3	300	10M+10F
4	1000	10M+10F

M = Males; F = Females

The test item was administered by oral gavage at a dose volume of 5 mL/kg.

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 32 days. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum or until the day before necropsy. The following investigations were performed on parental animals of all groups: body weight, clinical signs, food consumption, oestrous cycle, mating performance, litter data, macroscopic observations, organ weights and histopathological examination of abnormalities. Histopathological examination of testes, epididymides and ovaries was performed only on control and high dose groups.

Mortality and fate of females

One control female and one low dose female were found dead. On the basis of the macroscopic and microscopic observations, the cause of deaths is attributable to a misdosing. One low dose female was not pregnant and one mid-dose female showed unilateral implantation. The number of females with live pups on Day 4 post partum was 9 in the control group, 8 in the low dose group and 10 in each of the mid- and high dose groups.

Clinical signs

As consequence of the colour of the test item blue staining of the bedding material and tail was noted in the animals of mid- and high dose groups.

Body weight and body weight gain

No signs of toxicological significance were seen in body weight or body weight gain during the study in treated animals compared to controls.

Food consumption

No effects on food consumption were seen.

Oestrus cycle, mating performance and reproductive parameters

No treatment-related anomalies were noted in the oestrus cycle of treated females when compared to controls. Pre-coital interval, copulation plugs, copulatory and fertility index were similar between treated and control animals.

Implantation, pre-birth loss data and gestation length

No differences were observed in treated and control groups for these parameters.

Litter data and sex ratio

Litter data and sex ratios at birth and on Day 4 post partum were unaffected by treatment.

Clinical signs of pups

Pre-weaning clinical signs did not show treatment-related effects.

Necropsy findings in pups

Necropsy findings in decedent pups and pups sacrificed on Day 4 post partum did not reveal any treatment-related effect.

Terminal body weight and organ weights

Terminal body weight and organ weights were unaffected by treatment in both sexes.

Macroscopic and microscopic observations including examination of spermatogenic cycle

The lesions detected in some animals were also noted in untreated laboratory rats; they were therefore considered to be incidental and/or spontaneous in origin. No treatment-related changes were seen in testis, epididymides (including spermatogenic cycle) and ovaries evaluated in high dose males or females receiving the test item.

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

Quality of whole database:

reliable without restriction - well performed GLP compliant OECD guideline study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Short description of key information:
Reproduction/Developmental Toxicity (oral):
An OECD test guideline (OECD 421) and GLP-compliant Reproduction/Developmental Toxicity Screening Test was performed with the test item (Solvent Blue 104). Groups of 10 male and 10 female Wistar rats received doses of 0, 100, 300 and 1000 mg/kg bw by daily gavage. No toxicologically significant changes were noted in any of the parameters investigated in this study.

An NOAEL for general toxicity of 1000 mg/kg/day was established. No test item-related effects were observed in males or females up to and including 1000 mg/kg bw/day, the highest dose level administered.

An NOAEL for reproduction and development of 1000 mg/kg/day was established. No test item-related effects on reproduction or development were observed up to and including 1000 mg/kg bw/day, the highest dose level administered.

Reproduction/Developmental Toxicity (dermal):
The substance is considered not to exert adverse effects.

Reproduction/Developmental Toxicity (inhalation):
The substance is considered not to exert adverse effects.

Justification for selection of Effect on fertility via oral route:
no alternative study available

Effects on developmental toxicity

Description of key information

Reproduction/Developmental Toxicity (oral):
An OECD test guideline (OECD 421) and GLP-compliant Reproduction/Developmental Toxicity Screening Test was performed with the test item (Solvent Blue 104). Groups of 10 male and 10 female Wistar rats received doses of 0, 100, 300 and 1000 mg/kg bw  by daily gavage. No toxicologically significant changes were noted in any of the parameters investigated in this study. 
An NOAEL for general toxicity of 1000 mg/kg/day was established. No test item-related effects were observed in males or females up to and including 1000 mg/kg bw/day, the highest dose level administered.
An NOAEL for reproduction and development of 1000 mg/kg/day was established. No test item-related effects on reproduction or development were observed up to and including 1000 mg/kg bw/day, the highest dose level administered.
Reproduction/Developmental Toxicity (dermal):
The substance is considered not to exert adverse effects.
Reproduction/Developmental Toxicity (inhalation):
The substance is considered not to exert adverse effects.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed GLP compliant OECD guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 421

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 Italy S.p.A., Calco (Lecco), Italy
- Age at study initiation: 8-9 weeks
- Weight at study initiation: (P) Males: 317-322g; Females: 210-216 g;
- Housing:
From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulphone solid bottomed cages measuring approximately 59.5x38x20 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During the mating period, animals were housed on the basis of one male to one female in clear polycarbonate cages measuring approxiamtely 43x27x18 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily.
The males were re-caged after mating as they were before mating.
After mating, the females were transferred to individual clear polycarbonate solid bottomed cages measuring approxiamtely 43x27x18 cm
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20-24
- Humidity (%):40-70
- Air changes (per hr):15-20
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From: To:05 July 2012-05 September 2012

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The required amount of the test item was suspended in the vehicle (corn oil), brought to the final volume appropriate for each concentration (concentrations of 20, 60 and 200 mg/mL) and kept under magnetic stirrer at room temperature prior to use and until the time of dosing of the last animal.
Concentrations were calculated and expressed in terms of test item as supplied

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable (content check and homogeneity) and the stability of formulation was satisfactory. Results of the analyses were within the limits of acceptance.
The stability was found to be 24 hours at room temperature in the concentration range of 20 to 200 mg/mL.
Samples of the formulations prepared on Day 1 and on Week 4 of the study were also analysed to check the concentration and homogeneity. Results of the analyses were within the limits of acceptance.
Chemical analyses were carried out by the Analytical Chemistry Department at RTC, using a validated spectrophotometric detection in the range from 1.0 to 250 mg/mL.
The software used for this activity was Empower Probuild No. 2154.

Details on mating procedure:

Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).
The female was paired with the same male until positive identification occurred.

Duration of treatment / exposure:

Males were dosed for 32 days (including 2 weeks prior to pairing during the pre pairing period and approximately 2 weeks post pairing). Females were dosed 2 weeks before pairing, during the pairing period, gestation period and up to Day 3 post partum.

Frequency of treatment:

Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter through the day before
necropsy.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Duration of test:

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 32 days.
Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum or until the day before necropsy.

Remarks:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:Dose levels had been selected in consultation with the Sponsor based on information from a previous non GLP compliant dose range finding study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:daily

DETAILED CLINICAL OBSERVATIONS: NO

BODY WEIGHT: Yes
- Time schedule: weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on post partum day 4
- Organs examined:Ovaries and uterus

Ovaries and uterine content:

a) number of visible implantation sites;
b) number of corpora lutea (pregnant animals).

Fetal examinations:

- External examinations of pups at day 4 post partum: Yes:
- Soft tissue examinations: : No
- Skeletal examinations: No
- Head examinations: No

Statistics:

Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.

The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test.

The criterion for statistical significance was p<0.05.

Indices:

Group mean values were calculated for all parameters. Data from females not pregnant were excluded from group mean calculations as considered appropriate by the Study Director.

The following reproductive indices were calculated:

Males

Copulatory Index (%) = (no. of animals mated x 100) / (no. of animals paired)

Fertility Index (%) = (no. of males which induced pregnancy x 100) / (no. of males paired)


Females

Copulatory Index (%) = (no. of animals mated x 100) / (no. of animals paired)

Fertility Index (%) = (no. of pregnant females x 100) / (no. of females paired)


Males and females

Copulatory Interval = Mean number of days between pairing and mating


Females

Pre-birth loss was calculated as a percentage from the formula:
(((No. of visible implantations) - (total litter size at birth)) x 100) / (No. of visible implantations)

Pup loss at birth was calculated as a percentage from the formula:
(((Total litter size) - (live litter size)) x 100) / (Total litter size)

Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula:
(((Total litter size at birth) - (live litter size at Day 4)) x 100) / (Total litter size at birth)

Sex ratios were calculated at birth and on Day 4 post partum and presented as the percentage of males per litter.

Historical control data:

not used since no effects

Details on maternal toxic effects:

Maternal toxic effects:no effects

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: External examinations of pups at day 4 post partum

Details on embryotoxic / teratogenic effects:
External examinations of pups at day 4 post partum revealed no effects.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Clinical signs, body weight and food consumption were unaffected by treatment in both sexes.
Reproductive parameters such as fertility index, pre-coital interval and copulatory index did not show significant differences in treated groups compared to controls.
Gestation length, litter data, sex ratios and implantation losses (pre and post) were unaffected by treatment.
At macroscopic and microscopic examination, no treatment related lesions were seen.
On the basis of the results obtained in this OECD Reproduction / Developmental Toxicity Screening Test, the maximum dosage of 1000 mg/kg/day of the test item was established as the NOAEL (No Observed Adverse Effect Level) for parental animals of both sexes (general toxicity and reproduction) and for the offspring (developmental toxicitxy).

Executive summary:

Study design

The toxic effects on rats after repeated dosing, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition, development of the concepts and early lactation were investigated.

The dosage groups were as follows:

Group Number	Treatment (mg/kg/day)	Number of animals
1	0	10M+10F
2	100	10M+10F
3	300	10M+10F
4	1000	10M+10F

M = Males; F = Females

The test item was administered by oral gavage at a dose volume of 5 mL/kg.

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 32 days. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum or until the day before necropsy. The following investigations were performed on parental animals of all groups: body weight, clinical signs, food consumption, oestrous cycle, mating performance, litter data, macroscopic observations, organ weights and histopathological examination of abnormalities. Histopathological examination of testes, epididymides and ovaries was performed only on control and high dose groups.

Mortality and fate of females

One control female and one low dose female were found dead. On the basis of the macroscopic and microscopic observations, the cause of deaths is attributable to a misdosing. One low dose female was not pregnant and one mid-dose female showed unilateral implantation. The number of females with live pups on Day 4 post partum was 9 in the control group, 8 in the low dose group and 10 in each of the mid- and high dose groups.

Clinical signs

As consequence of the colour of the test item blue staining of the bedding material and tail was noted in the animals of mid- and high dose groups.

Body weight and body weight gain

No signs of toxicological significance were seen in body weight or body weight gain during the study in treated animals compared to controls.

Food consumption

No effects on food consumption were seen.

Oestrus cycle, mating performance and reproductive parameters

No treatment-related anomalies were noted in the oestrus cycle of treated females when compared to controls. Pre-coital interval, copulation plugs, copulatory and fertility index were similar between treated and control animals.

Implantation, pre-birth loss data and gestation length

No differences were observed in treated and control groups for these parameters.

Litter data and sex ratio

Litter data and sex ratios at birth and on Day 4 post partum were unaffected by treatment.

Clinical signs of pups

Pre-weaning clinical signs did not show treatment-related effects.

Necropsy findings in pups

Necropsy findings in decedent pups and pups sacrificed on Day 4 post partum did not reveal any treatment-related effect.

Terminal body weight and organ weights

Terminal body weight and organ weights were unaffected by treatment in both sexes.

Macroscopic and microscopic observations including examination of spermatogenic cycle

The lesions detected in some animals were also noted in untreated laboratory rats; they were therefore considered to be incidental and/or spontaneous in origin. No treatment-related changes were seen in testis, epididymides (including spermatogenic cycle) and ovaries evaluated in high dose males or females receiving the test item.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable without restriction - well performed GLP compliant OECD guideline study

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

Justification for selection of Effect on developmental toxicity: via oral route:
no alternative study available

Justification for classification or non-classification

Solvent Blue 104 does not have to be classified regarding reproductive toxicity according to the criteria laid down in Directive 67/548/EEC and in Regulation (EC) No 1272/2008, because:

- Solvent Blue 104 caused no relevant systemic effects and no effects related to reproduction and development when tested in a Reproduction/Developmental Toxicity Screening Test. In this study oral application of Solvent Blue 104 revealed NOAEL's of 1000 mg/kg/day for general toxicity and for reproduction and development respectively.

- Solvent Blue 104 caused no relevant systemic effects and particularly no effects with regard to the reproductive organs when tested in a Repeated Dose Toxicity Study. In this study oral application of Solvent Blue 104 revealed an NOAEL of 1000 mg/kg/day.

 

It can reasonably be deduced that Solvent Blue 104 does not exert reproductive toxicity after dermal application and thus does not have to be classified according to the criteria laid down in Directive 67/548/EEC and in Regulation (EC) No 1272/2008 and that testing is not scientifically necessary, because:

- Solvent Blue 104 caused no relevant systemic effects and no effects related to reproduction and development when tested in a Reproduction/Developmental Toxicity Screening Test. In this study oral application of Solvent Blue 104 revealed NOAEL's of 1000 mg/kg/day for general toxicity and for reproduction and development respectively.

- Solvent Blue 104 caused no relevant systemic effects and particularly no effects with regard to the reproductive organs when tested in a Repeated Dose Toxicity Study. In this study oral application of Solvent Blue 104 revealed an NOAEL of 1000 mg/kg/day.

- It is unlikely that Solvent Blue 104 becomes systemically bioavailable to a relevant extend after dermal exposure due to its low solubility in water and in n-octanol.

- Repeated dose toxicity after dermal application is not considered to be higher than after oral administration.

- Testing for acute dermal toxicity revealed no signs of bioavailability or toxicity.

- Solvent Blue 104 does not have to be classified as skin sensitizing or as skin or eye irritating, indicating that its chemical inertness and low solubility in water and n-octanol largely prevent interaction with living cells and tissues.

 

It can reasonably be deduced that Solvent Blue 104 does not exert reproductive toxicity after inhalation and thus does not have to be classified according to the criteria laid down in Directive 67/548/EEC and in Regulation (EC) No 1272/2008 and that testing is not scientifically necessary, because:

- Solvent Blue 104 caused no relevant systemic effects and no effects related to reproduction and development when tested in a Reproduction/Developmental Toxicity Screening Test. In this study oral application of Solvent Blue 104 revealed NOAEL's of 1000 mg/kg/day for general toxicity and for reproduction and development respectively.

- Solvent Blue 104 caused no relevant systemic effects and particularly no effects with regard to the reproductive organs when tested in a Repeated Dose Toxicity Study. In this study oral application of Solvent Blue 104 revealed an NOAEL of 1000 mg/kg/day.

- It is unlikely that Solvent Blue 104 becomes systemically bioavailable to a relevant extend after inhalation due to its low solubility in water and in n-octanol.

- Solvent Blue 104 does not have to be classified as skin sensitizing or as skin or eye irritating, indicating that its chemical inertness and low solubility in water and n-octanol largely prevent interaction with living cells and tissues.

- Solvent Blue 104, when aerosolized in respirable form, is likely to behave like an inert dust.