Chloro[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]aluminium

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Chloro[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]aluminium

EC Number:

237-998-4

EC Name:

Chloro[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]aluminium

Cas Number:

14154-42-8

Molecular formula:

C32H16AlClN8

IUPAC Name:

31h-phthalocyaninato(2-)-n29,n39,n31,n32]-chloro[29(sp-5-12)-aluminu; chloro[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-,(SP-5-12)-Aluminum;CHLOROALUMINUM PHTHALOCYANINE;CHLORO(29H,31H-PHTHALOCYANINATO)ALUMINUM; CHLORO(PHTHALOCYANINATO)ALUMINIUM;ALUMINUM PHTHALOCYANINE CHLORIDE;PHTHALOCYANINE CHLOROALUMINUM; chloro[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]aluminium

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Species/Strain: CRL Sprague-Dawley CD® IGS rats
The Sprague-Dawley® rat is the system of choice because, historically, it has been a preferred and commonly used species for developmental and reproductive toxicity studies. The current state of scientific knowledge does not provide acceptable alternatives to the use of live animals to accomplish the objective of this study. Because of extensive reporting in the literature, this strain provides a historical reference point to aid in interpretation of the study data.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Number of Animals: 104 (40 virgin male and 64 virgin female rats)
Number of Groups: 8 (2 cohorts of 3 dose levels per sex + 1 control group per sex)
Number of Animals per Group: 10 males and 11 females in Reproduction/Developmental cohort (Groups 1-4), and 5 females in General Toxicity cohort (Groups 5-8).
Species/Strain: CRL Sprague-Dawley CD® IGS rats
Body Weight, Age, and Sex: Male and female rats were ordered from the supplier on the basis of age and birth date. Male and female rats were from different barriers in order to avoid brother-sister mating. Male and female rats were approximately 9 weeks of age at the time of arrival at PSL. The weight variation did not exceed ± 20% of the mean weight.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected by the Sponsor. This route of administration is recommended in the referenced guidelines (Section 10.C) and a potential route of human exposure.

Vehicle:

water

Remarks:

0.5% Carboxymethylcellulose in water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose levels of 0 (vehicle control), 250, 500, and 1000 mg/kg/day of C.I. Pigment Blue 79 were selected by the Sponsor in consultation with the Study Director. The dose levels were based on the results of a previous range-finding/toxicity study (PSL, draft). The high dose was the maximum recommended dose based on referenced guidelines (Section 10.C) and was not expected to cause marked toxicity. The low and intermediate dose levels were selected to derive a dose-response for any effects observed. A NOAEL was expected to be achieved for this study.

Group 1-4 females began this study with a 14-day pre-dose estrous assessment. After the estrus assessment, Groups 1-4 males and females were dosed while housed separately for a 14-day premating period, followed by dosing through a 14-day co-habitation period. Upon determination of pregnancy, the females were removed to a separate cage and continued to be dosed through pregnancy and lactation until terminal sacrifice on lactation day (LD) 14, whereas the males were dosed until terminal sacrifice after at least 28 days of administration on Day 47. Group 5-8 females were dosed concurrently with the Groups 1-4 males until terminal sacrifice after at least 28 days of administration on Day 47.

Duration of treatment / exposure:

Each animal was dosed by oral intubation using a stainless steel ball-tipped gavage needle attached to an appropriate syringe. Dose administration was daily (7 days/week) for all adult animals as follows:
 Group 1-4 male rats were dosed during pre-mating (14 days) and mating/post-mating (14 days) periods, for at least 28 days of administration.
 Group 1-4 female rats in the - Reproduction/Developmental Toxicity Cohort were dosed during pre-mating (14 days), mating (up to 14 days), gestation (approximately 22 days), and lactation (13 days) periods.
 Group 5-8 female rats in the General Toxicity Cohort were dosed for at least 28 days of administration.

The dose mixtures were maintained on a magnetic stir plate during dose administration. The first day of administration was Day 15 of the study. Dosing was at approximately the same time each day (±2 hours) with an exception on the day(s) hematology and/or clinical chemistry samples were collected. Residual dose mixtures were properly discarded following daily administration and sampling (as required).

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1
REPRODUCTION/DEVELOPMENTAL TOXICITY COHORT
No. Animals/Group: 10M / 11F
Vehicle Control

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Group 2
REPRODUCTION/DEVELOPMENTAL TOXICITY COHORT
No. Animals/Group: 10M / 11F
Low Dose

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Group 3
REPRODUCTION/DEVELOPMENTAL TOXICITY COHORT
No. Animals/Group: 10M / 11F
Intermediate Dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4
REPRODUCTION/DEVELOPMENTAL TOXICITY COHORT
No. Animals/Group: 10M / 11F
High Dose

Doses / concentrations 5

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 5
GENERAL TOXICITY COHORT
No. Animals/Group: 5F
Vehicle Control

Doses / concentrations 6

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Group 6
GENERAL TOXICITY COHORT
No. Animals/Group: 5F
Low Dose

Doses / concentrations 7

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Group 7
GENERAL TOXICITY COHORT
No. Animals/Group: 5F
Intermediate Dose

Doses / concentrations 8

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 8
GENERAL TOXICITY COHORT
No. Animals/Group: 5F
High Dose

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

All animals were observed at least twice daily for viability

Sacrifice and pathology:

Clinical Pathology
Clinical pathology was performed on five males (last 5 males assigned to Groups 1-4) and five females (Groups 5-8) at each dose level near the end of their test period. The animals were fasted overnight (at least 15 hours) prior to each blood collection. Blood samples for hematology (except coagulation samples) and clinical chemistry were collected via sublingual bleeding under isoflurane anesthesia. Approximately 1000 μL was collected in a precalibrated tube containing K2EDTA for hematology assessments. The whole blood samples were stored under refrigeration until analysis. Approximately 1000 μL was collected into a tube containing no preservative for clinical chemistry assessments. These samples were spun in a refrigerated centrifuge, and the serum was transferred to a labeled tube. Serum samples were stored in a -80ºC freezer until analysis.
Blood samples used to determine the prothrombin time and activated partial thromboplastin time (coagulation) were collected via the inferior vena cava under isoflurane anesthesia at terminal sacrifice. Approximately 1.8 mL of blood was collected in a pre-calibrated tube containing 3.2% sodium citrate. These samples were centrifuged in a refrigerated centrifuge and the plasma was transferred to labeled tubes. Plasma samples were stored in a -80ºC freezer until analysis.
In addition, a blood sample for serum was collected from all animals (fasted overnight prior toterminal sacrifice) via sublingual bleeding under isoflurane anesthesia and retained for possible future evaluation, if treatment-related effects were identified. Details of this evaluation were added by amendment, if applicable. If no further evaluation was warranted by treatment-related, this sample was discarded upon finalization of the study.
All blood samples were evaluated for quality by visual examination.

Terminal Sacrifice and Histopathology
1 Scheduled Sacrifice
All Groups 1-4 female rats (with or without surviving pups) were sacrificed on LD 14. All surviving Groups 1-4 male rats and Groups 5-8 female rats will be sacrificed following at least 28 days of test substance administration. Two females (pregnant or non-pregnant) that did not deliver a litter were sacrificed 24-26 days after the last day of possible mating or GD25 provided the animal’s weight, palpation of the abdomen, or other signs that do not suggest pending parturition.
All animals were fasted overnight prior to terminal sacrifice. At terminal sacrifice, the animals were euthanized by exsanguination from the abdominal aorta under isoflurane anesthesia. A gross necropsy of all males and females (including decedents) included examination of the external surface of the body, all orifices, musculoskeletal system, and the cranial, thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Special attention was paid to the organs of the reproductive system. All abnormalities, including gross lesions were recorded.
2 Histopathology
Histopathology examination was performed on the preserved organs and tissues of the Reproductive/Developmental Toxicity and General Toxicity animals from the control and high dose groups (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure). The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy. Slide preparation and histopathological assessment will be performed by a board-certified veterinary pathologist at Histo-Scientific Research Laboratories (HSRL).

Other examinations:

A. Clinical Observations
All animals were observed at least twice daily for viability. Cage-side observations of all animals were performed daily during the acclimation, pre-mating, mating, gestation, and lactation periods with the exception of one Group 3 female (Animal 8035) that was assessed on GD 9 rather than GD 7 (Deviation 1). All observations were recorded.
Prior to the first treatment with the test substance, a detailed observation was conducted. Detailed observations were conducted weekly thereafter during the pre-mating, mating, and post-mating periods (as applicable) for males and females, and approximately weekly for the females during gestation and lactation periods. These observations were conducted both while handling the animal and with the animal placed in an open field. Potential signs noted included, but were not limited to: changes in skin, fur, eyes, and mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Likewise, changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling), difficult or prolonged parturition, or bizarre behavior (e.g., self-mutilation, walking backwards) was also recorded.
Toward the end of the gestation period, pregnant females were examined twice daily for signs of parturition. The females were allowed to give birth (F1), and the gestation length was calculated. The females were evaluated for adverse clinical signs during parturition. Maternal behavior (e.g., nursing, handling of pups, etc.) was checked on LD 0, LD 4, and LD 13, with any abnormal behavior recorded.
A palpable mass was observed for Animal 8017 and was tracked on a regular basis after discovery.

B. Estrous Cycles
Estrous cycles were monitored for all Groups 1-4 females, daily for the 14-day pre-dose period, as well as during the 14-day pre-mating and mating periods until evidence of mating, by vaginal smears to evaluate for regular cyclicity. A vaginal smear was also collected from these females at termination to determine the stage of estrous at terminal sacrifice.

C. Functional Observation Battery
A functional observational battery (FOB) was performed once after the end of the mating period on Day 46 using five males (last 5 males assigned to Groups 1-4) and five females (Groups 5-8) at each dose level including controls. Each rat was evaluated during handling and while in an open field for excitability, autonomic function, gait and sensorimotor coordination (open field and manipulative evaluations), reactivity and sensitivity (elicited behavior), and other abnormal clinical signs including but not limited to convulsions, tremors, unusual or bizarre behavior, emaciation, dehydration and general appearance. The rats were observed in random order and without the observer having knowledge of the treatment group.
In addition to the above observations, forelimb and hindlimb grip strength and foot splay measurements were obtained and recorded. The grip strength was measured with a digital force gauge (Dillion GS series). Triplicate measurements of grip strength and duplicate measurements for foot splay were taken for each animal and a mean was calculated for each mesurement.

D. Motor Activity
Motor activity (MA) was also evaluated on Day 46 on the five males (last 5 males assigned to Groups 1-4) and five females (Groups 5-8) at each dose level including controls assigned for the FOB evaluation. This assessment took place at the same period during the study as the FOB. Activity was monitored using an automated Photobeam Activity System®, San Diego Instruments, Inc. The system could monitor up to twenty animals during one session. An equal number of animals assigned for MA assessment from each dose level were evaluated in each session, if applicable. Each animal was placed into a polycarbonate solid bottom cage and the evaluation phase began after the room was darkened and steps were taken to avoid excess noise that may affect the animal’s behavior. Each animal was evaluated for a single one-hour phase, with photobeam counts accumulated over six 10-minute intervals.

E. Body Weight and Body Weight Gain
E.1 Male Rats
All male rats were weighed weekly during the pre-mating and mating periods, and prior to terminal sacrifice.
E.2 Female Rats
All female rats were weighed weekly during pre-mating, mating, and gestation periods, within 24 hours of parturition (i.e., LD 0 or LD 1) and LD 7, and prior to terminal sacrifice on LD 14, as applicable, with the exception of one Group 3 female (Animal 8035) that was assessed on GD 9 rather than GD 7 (Deviation 1-3). Females showing no evidence of mating were assigned GD 0 after cessation of cohabitation and body weights were measured weekly. Body weight gain was calculated for selected intervals and for the study overall.

F. Food Consumption and Food Efficiency
Individual food consumption was measured and recorded to coincide with body weight measurements for all animals of the throughout the study. Food consumption was not measured during cohabitation. During cohabitation (mating period), when two rats occupied the same cage with one feed jar, replenishment of the feed jars was documented, but actual values were not recorded. Food efficiency was calculated and reported for all applicable intervals.

G. Mating
After 14 days of treatment, animals were randomly mated by placing one female in the breeding cage of a male from the same dose group. A record of mating pairs was maintained. The cohabitation period consisted of a maximum of 14 days. Vaginal smears were performed daily on females during the mating period, and the presence or absence of sperm or vaginal plug was recorded. Females considered to have successfully mated (determined by the presence of vaginal sperm and/or a vaginal copulation plug) were removed from the cage of the male, assigned a GD 0 and housed individually. Female rats that had not mated within the first seven days of cohabitation were randomly assigned and paired with alternate male rats that have successfully mated. These female rats remained in cohabitation with the second set of males for a maximum of seven additional days. If successful mating had not occurred after 14 days of cohabitation, females were assigned a GD 0 on the morning of the last day of the cohabitation period and were evaluated as if in gestation thereafter. After the mating period, any female with no positive evidence of mating was individually housed in a plastic solid bottom polycarbonate shoebox cage containing nesting material.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no mortalities over the course of the study. There were no adverse clinical or detail observations attributable to the administration of C.I. Pigment Blue 79. Clinical observations of blue stainging or blue discoloration of feces observed in the treated male and female rats were secondary to test substance administration and are considered to have no toxicological relevance.

Mortality:

no mortality observed

Description (incidence):

There were no mortalities over the course of the study. There were no adverse clinical or detail observations attributable to the administration of C.I. Pigment Blue 79. Clinical observations of blue stainging or blue discoloration of feces observed in the treated male and female rats were secondary to test substance administration and are considered to have no toxicological relevance.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes in male or female body weight endpoints attributable to the administration of C.I. Pigment Blue 79.
Mean weekly body weights and daily body weight gains for the test substancetreated males and female rats at 250, 500, and 1000 mg/kg/day of C.I. Pigment Blue 79 for the Reproduction/Developmental Toxicity Cohort and General Toxicity Cohort were comparable to control values throughout the study. Mean weekly body weights and daily body weight gains for the test substance-treated females during gestation and lactation periods showed a steady increase appropriate to their physiologic state, were within normal range to that expected for this age and strain of rat, and were comparable to control values throughout the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no changes in male and female mean daily food consumption or food efficiency attributable to the administration of C.I. Pigment Blue 79.
Mean daily food consumption and food efficiency for the test substance-treated males and female rats in administered 250, 500, and 1000 mg/kg/day of C.I. Pigment Blue 79 for the Reproduction/Developmental Toxicity Cohort and General Toxicity Cohort were comparable to the control values throughout the study.

Food efficiency:

no effects observed

Description (incidence and severity):

There were no changes in male and female mean daily food consumption or food efficiency attributable to the administration of C.I. Pigment Blue 79.
Mean daily food consumption and food efficiency for the test substance-treated males and female rats in administered 250, 500, and 1000 mg/kg/day of C.I. Pigment Blue 79 for the Reproduction/Developmental Toxicity Cohort and General Toxicity Cohort were comparable to the control values throughout the study.

Water consumption and compound intake (if drinking water study):

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no adverse functional observation battery findings attributed to the administration of C.I. Pigment Blue 79. Mean quantitative measurements of grip strength and foot splay for the test substance-treated male and female rats were comparable to the respective vehicle control group values.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test substance-related organ weight changes attributable to the administration of C.I. Pigment Blue 79.
A significant decrease (p<0.05) in heart weight for Group 3 males (general toxicity cohort) and significant decreases (p<0.05-0.01) in heart-to-brain weight ratios in Group 3 and 4 males (general toxicity cohort) were observed that, without correlating significant changes in absolute organ weights and relative organ-to-body or associated microscopic findings, was interpreted to be incidental and not toxicologically relevant.
A significant increase (p<0.05) in oviducts-to-brain weight ratio was observed for Group 6 and 8 females that, without correlating significant changes in absolute organ weights and relative organ-to-body or associated microscopic findings, was interpreted to be incidental and not toxicological relevant.
All other absolute and relative organ weights for the adult treated animals at 250, 500, and 1000 mg/kg/day of C.I. Pigment Blue 79 were comparable with their respective control values.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related changes in hematology, coagulation andclinical chemistry parameters attributable to the administration of C.I. Pigment Blue 79.
Hematology: There were no test substance-related changes in hematology parameters on Day 45.
Coagulation: There were no test substance-related changes in coagulation parameters on Day 47.
Clinical Chemistry: There were no test substance-related changes in serum chemistry values on Day 45.

Effect levels

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for reproductive performance and general toxicological assessment of the test substance, C.I. Pigment Blue 79, was determined to be 1000 mg/kg/day for male and female Sprague-Dawley rats.

Executive summary:

A combined repeated oral dose gavage toxicity study with reproduction/developmental screening was conducted in Crl: CD® (SD) IGS rats to provide information on male and female general toxicity and reproductive performance, including gonadal function, mating behavior, conception, development of conceptus’, and parturition and lactation following administration of C.I. Pigment Blue 79.

One hundred-four healthy rats (40 virgin male and 64 virgin female) were selected for the test and distributed into four Reproduction/Developmental Toxicity Cohort groups (Groups 1-4; 10 males and 11 females per group) and four General Toxicity Cohort groups (Groups 5-8; 5 females/dose level). Dose levels of 250, 500, and 1000 mg/kg/day of C.I. Pigment Blue 79 for Groups 2-4 and 6-8, respectively, as well as a vehicle control (Groups 1 and 5; 0.5% carboxymethylcellulose (CMC) in water), were selected for the test.

Males and female rats were housed separately for a 14-day pre-dosing, followed by a 14-day premating period. The males and females received the test substance while housed separately for the 14-day pre-mating period, followed by dosing through a 14-day co-habitation period. Estrus cycles were monitored daily for all females in Groups 1-4 during the pre-dosing, pre-mating, and mating periods until evidence of mating. Upon determination of pregnancy or following the prescribed 14-day mating period, females were removed and placed in a separate cage, where dosing continued through the gestation period of pregnancy until Day 13 of lactation. Males were sacrificed following the gestation phase (Day 47), and the first five males in Groups 1-4 were evaluated for fertility, including spermatogenic stage and epididymal effects. The last five males assigned to Groups 1-4 were selected for general toxicity evaluations, along with the five females in Groups 5-8. Blood was sampled from the last five Groups 1-4 males and five Groups 5-8

females on Study Day 45 for hematology and clinical chemistry, and just prior to terminal sacrifice on Day 47 for coagulation assessments. Gross necropsies were performed on all study animals, and the males and females were evaluated histopathologically according to protocol design following scheduled sacrifice on Study Day 47 (Group 1-4 males and Group 5-8 females) and LD14 (Group 1-4 females).

The animals were observed for viability, signs of gross toxicity, and behavioral changes at least once daily during the study, and approximately weekly for a battery of detailed observations (beginning on Day 15 to coincide with initiation of dosing). A Functional Observational Battery (FOB) and a Motor Activity (MA) test were performed on the selected animals (last five Groups 1-4 males and five Groups 5-8 females) on Study Day 45. Individual body weights and food consumption were recorded weekly for both sets of animals. Body weights for females were recorded on GD 0, 7, 14, and 21, as well as on LD 0, 7, and 14. Food consumption for males and females was recorded to coincide with body weight collection exception during the mating period.

The neat test substance was considered to be stable throughout the duration of the study. All dose preparations were homogeneously distributed. In general, the concentration verification results were consistent with the targeted concentrations of 25, 50, and 100 mg/mL.

There were no mortalities over the course of the study. There were no adverse clinical or detail observations attributable to the administration of C.I. Pigment Blue 79. The number of estrus cycles was comparable between the control and test substance-treated females during the predosing and pre-mating periods.

There were no adverse functional observation battery findings attributed to the administration of C.I. Pigment Blue 79. Mean quantitative measurements of grip strength and foot splay as well as motor activity measurements for the test substance-treated male and female rats were comparable to the respective vehicle control group values.

There were no test substance-related changes in mean weekly body weights, daily body weight gain, daily food consumption, or food efficiency for the test substance-treated male and female rats throughout the study.

There were no test substance-related changes in hematology, coagulation and clinical chemistry parameters in test substance-treated male and female rats.

There were no adverse macroscopic findings attributable to the administration of C.I. Pigment Blue 79. Blue tissue discoloration, attributed to the blue dye test substance, was primarily restricted to the gastrointestinal tract and mesenteric lymph nodes and interpreted to be of no toxicological significance as there were no correlating microscopic findings. There were no reproductive or general toxicologic microscopic changes related to C.I. Pigment Blue 79 administration. No test substance-related organ weight changes were found in the test substancetreated male or female rats.

There were no changes in male or female mating, fertility, and fecundity indices attributable to the administration of C.I. Pigment Blue 79. C.I. Pigment Blue 79 administration resulted in no significant changes in any of the gestational or birth parameters evaluated during the course of the present study including gestational length, gestation index, number of implantation sites, corpora lutea, average resorption (early) numbers and pre-implantation and post-implantation loss. In addition, C.I. Pigment Blue 79 administration

resulted no significant differences in earlier stage of gestation indicators such as newborn pup litter size, live birth index, and number of stillborn pups and sex ratio.

In the offspring generation (F1), there were no adverse clinical observations attributable to the administration of C.I. Pigment Blue 79. There were no test substance-related changes in the average pup body weight and body weight gains per litter. There were no adverse necropsy findings in the F1 pups attributed to the test substance administration.

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for reproductive performance and general toxicological assessment of the test substance, C.I.

Pigment Blue 79, was determined to be 1000 mg/kg/day for male and female Sprague-Dawley rats.