Hydrogen [29H,31H-phthalocyaninesulphonato(3-)-N29,N30,N31,N32]cuprate(1-), compound with dodecylamine (1:1)

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:

2014-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD guideline compliant study with well-characterized test material

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France or Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation:
- Weight at study initiation:
- Fasting period before study:
- Housing:
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 - 70%
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 8 Dec 2014 To: 30 Jan 2015

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) are be prepared daily within 6 hours prior to dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item is insoluble in water, but suspendible in propylene glycol
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal
copulatory plug. This day was designated Day 0 post-coitum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

28 days (males), ca 53 days (females)

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
100, 300 and 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: Range-finding standy
- Rationale for animal assignment (if not random): Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: mortality, clinical observations

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first exposure and at the end of the study as part of the functional observation battery (FOB)

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of male and female animals were recorded shortly before the start of
administration of the test item at randomization and at the start of the study (day 0). Males
were weighed weekly until sacrifice. Females were weighed once per week during the premating period. Mated females were
weighed on days 0, 7, 14 and 21 during presumed gestation and on day 0 and 4 of lactation.
Non-mated females were weighed once per week after the mating period. In addition, the
animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.


FOOD CONSUMPTION:
Feed consumption was measured per cage over weekly intervals during the study, with exemption of the mating period, during which no feed consumption was registered.


HAEMATOLOGY
Prior to sacrifice, 5 animals/sex/group were fasted overnight (water was freely available) and blood was taken from the aorta during necropsy, whilst under CO2/O2 anaesthesia. K3-EDTA was used as anticoagulant. In each sample the following determinations were carried out:
haemoglobin
packed cell volume
red blood cell count
reticulocytes
total white blood cell count
differential white blood cell counts (neutrophils, lymphocytes, eosinophils,
basophils, monocytes)
prothrombin time
thrombocyte count
mean corpuscular volume (MCV; calculated)
mean corpuscular haemoglobin (MCH; calculated)
mean corpuscular haemoglobin concentration (MCHC; calculated)
activated partial prothoplastin time (APTT)
red blood cell districution width (RDW)

CLINICAL CHEMISTRY
Prior to sacrifice, 5 animals/sex/group were fasted overnight (water was freely available) and blood was taken from the aorta during necropsy, whilst CO2/O2 anaesthesia. Blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. The following measurements were made in the plasma:
alkaline phosphatase activity (ALP),
aspartate aminotransferase activity (ASAT),
alanine aminotransferase activity (ALAT),
gamma glutamyl transferase activity (GGT),
total protein,
albumin,
ratio albumin to globulin (calculated),
urea,
creatinine,
glucose (fasting),
bilirubin (total),
cholesterol (total),
triglycerides,
phospholipids,
calcium (Ca),
sodium (Na),
potassium (K),
chloride (Cl),
inorganic phosphate (PO4),
bile acids


NEURO-BEHAVIOURAL TESTING (FOB) AND SPONTANEOUS MOTOR ACTIVITY
FOB and spontaneous motor activity were assessed in all study animals during the predose phase and in 5 animals/sex/group at the end of the study.


Sacrifice and pathology
GROSS NECROPSY AND HISTOLOGY OF PARENTAL ANIMALS
All surviving male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. Male animals were sacrificed after the mating period. Female animals were sacrificed at or shortly after day 4 of lactation. A necropsy was performed on animals that died intercurrently (if not precluded by autolysis) or that had to be killed because they were moribund. Prior to preservation of organs/tissues, the following organ weights were recorded: adrenals, brain, heart, kidneys, liver, ovaries, prostate, seminal vesicles, spleen, thymus, thyroid, uterus. Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which were preserved in Bouin's fixative:

- ovaries (after counting the corpora lutea)
- uterus (after counting of the implantation sites)
- testes
- epididymides
- seminal vesicles
- prostate
- all gross lesions

In addition of 5 animals/sex/group following organs were preserved:
adrenals
bone marrow (femur)
brain (including sections of cerebrum, cerebellum, medulla/pons)
caecum
cervix
clitorial gland
colon
coagulation gland
duodenum
eyes
femur including joint
heart
ileum
jejunum (including Peyer's patches)
kidneys
liver
lungs
mesenterial and axillary lymph nodes
ovaries
peripheral nerve (sciatic or tibial)
pituitary gland
preputial gland
prostate
rectum
seminal vesicles (including coagulation gland)
skeletal muscle (thigh)
spinal cord (cervical, mid-thoracic and lumbar)
spleen
stomach*
thymus
thyroid (including parathyroid)
trachea
urinary bladder
uterus
vagina
* Non glandular (“forestomach”) and glandular (fundus, pylorus) parts were examined
microscopically.

Microscopic examination was performed on the collected organs of all animals of the control (group 1) and high dose group (group 4). If treatment-related changes were observed in the high-dose group, the evaluation of these tissues/organs was extended to the intermediate-dose groups (2 and 3).
In addition, reproductive organs of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined. Furthermore, organs showing gross lesions of animals of all groups were microscopically examined.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testes weight, epididymis weight, spermatogenesis.
Slides of the testes were made of all males of Groups 1 and 4 and stained with PAS/haematoxylin to assess spermatogenesis.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

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

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals [day 28, details see entries for repeated dose toxicity]
- Maternal animals: All surviving animals [between postnatal days 4 and 7]
GROSS NECROPSY AND HISTOLOGY OF PARENTAL ANIMALS
All surviving male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. Male animals were sacrificed after the mating period. Female animals were sacrificed at or shortly after day 4 of lactation. A necropsy was performed on animals that died intercurrently (if not precluded by autolysis) or that had to be killed because they were moribund. Prior to preservation of organs/tissues, the following organ weights were recorded: adrenals, brain, heart, kidneys, liver, ovaries, prostate, seminal vesicles, spleen, thymus, thyroid, uterus. Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which were preserved in Bouin's fixative:

- ovaries (after counting the corpora lutea)
- uterus (after counting of the implantation sites)
- testes
- epididymides
- seminal vesicles
- prostate
- all gross lesions
In addition of 5 animals/sex/group following organs were preserved:
adrenals
bone marrow (femur)
brain (including sections of cerebrum, cerebellum, medulla/pons)
caecum
cervix
clitorial gland
colon
coagulation gland
duodenum
eyes
femur including joint
heart
ileum
jejunum (including Peyer's patches)
kidneys
liver
lungs
mesenterial and axillary lymph nodes
ovaries
peripheral nerve (sciatic or tibial)
pituitary gland
preputial gland
prostate
rectum
seminal vesicles (including coagulation gland)
skeletal muscle (thigh)
spinal cord (cervical, mid-thoracic and lumbar)
spleen
stomach*
thymus
thyroid (including parathyroid)
trachea
urinary bladder
uterus
vagina
* Non glandular (“forestomach”) and glandular (fundus, pylorus) parts were examined
microscopically.

Microscopic examination was performed on the collected organs of all animals of the control (group 1) and high dose group (group 4). If treatment-related changes were observed in the high-dose group, the evaluation of these tissues/organs was extended to the intermediate-dose groups (2 and 3).
In addition, reproductive organs of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined. Furthermore, organs showing gross lesions of animals of all groups were microscopically examined.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 5- 7 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

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

Descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Statistics:

Statistics
If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-toone t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Reproductive indices:

Mating index (%) Number of females mated x 100 / Number of females paired

Fertility index (%) Number of pregnant females x 100 / Number of females paired

Conception index (%) Number of pregnant females x 100 / Number of females mated

Gestation index (%) Number of females bearing live pups x 100 / Number of pregnant females

Duration of gestation Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Percentage live females at first litter check: Number of live female pups at First Litter Check x 100 Number of live pups at First Litter
Check

Percentage live males at first litter check: Number of live male pups at First Litter Check x 100 / Number of live pups at First Litter Check

Percentage of postnatal loss: Number of dead pups before planned necropsy x 100 / Number of live pups at First Litter Check

Viability index: Number of live pups before planned necropsy x 100 / Number of pups born alive

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:

effects observed, treatment-related

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and spermatogenic staging profiles were normal for all males examined (except male no. 15 of the low dose group).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.
The numbers of pregnant females were 10, 9, 9 and 10 at 0, 100, 300 and 1000 mg/kg bw/day, respectively. The numbers of females with living pups on Day 1 of lactation at these dose levels were 9, 9, 9 and 10, respectively.

Dose descriptor:

NOEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Histopathology findings in liver, kidney, mesenteric lymph nodes, adrenal glands at 1000 mg/kg bw

Dose descriptor:

NOEL

Remarks:

Fertility

Effect level:

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

Based on:

test mat.

Sex:

male/female

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

The number of dead and living pups at first litter check, postnatal loss and viability index were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings

At 1000 mg/kg bw/day, the sex ratio of the pups indicated more male than female pups (61% male pups versus 47% in the control group). However, the sex ratio was within the range observed in control groups of the laboratory (up to 64% males).
The mean numbers of living pups at first litter check were slightly lower at 300 and 1000 mg/kg bw/day than in the control group. There was no dose-related response and the values were within normal limits (Historical control data for number of living pups at first litter check for rats of the strain and age used in this study: mean of 1083 litters = 11.7; P5 = 7.0; P95 = 15.0). Moreover, the lower mean values were particularly due to one or two small litters in these test groups (four pups in litter no. 63 and one pup in litter no. 66 at 300 mg/kg bw/day; two pups in litter no. 72 at 1000 mg/kg bw/day). Therefore, these findings were considered not to be related to treatment.

At the first litter check, two pups of the contol group, one pup at 100 mg/kg bw/day and one pup at 300 mg/kg bw/day were found dead. During lactation, three pups of the control group, one pup at 100 mg/kg bw/day and 1 pup at 300 mg/kg bw/day went missing, and one pup at 1000 mg/kg bw/day was found dead. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Dose descriptor:

NOEL

Remarks:

development

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Exposure via treatment of dams until PND4.

Reproductive effects observed:

not specified

Table 1: Fertility indeces

	control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Mating index (%) (Females mated / Females paired) * 100	100	90	100	100
Fertility index (%) (Pregnant females / Females paired) * 100	100	90	90	100
Conception index (%) (Pregnant females / Females mated) * 100	100	100	90	100
Gestation index (%) (Females with living pups on Day 1 / Pregnant females) * 100	90	100	100	100

Table 2: Female fertility parameters

control

100 mg/kg bw

300 mg/kg bw

1000 mg/k

Corpus Lutea	MEAN	13.9	14.9	12.0	12.7
	ST.DEV	3.8	2.6	3.8	4.3
	N	10	9	9	10
Implantations	MEAN	12.2	13.0	10.9	11.6
	ST.DEV	4.3	2.1	4.8	3.7
	N	10	9	9	10

Conclusions:

No adverse effects on development and fertility were noted in the screening study in rats at dose levels of 100, 300 and 1000 mg/kg bw (OECD 422, GLP).

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:

valid without restriction

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/day (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, propylene glycol, alone. Males were exposed for 2 weeks prior to mating, during mating, and up to termination for 29 days. The females were exposed for 2 weeks prior to mating, during mating, duringpost-coitum, and at least 4 days of lactation (total exposure period 42-56 days).

 

Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 5 hours at room temperature.

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day).

 No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, numbers of corpora lutea and implantation sites, spermatogenic profiling, and histopathological examination of reproductive organs).

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e.gestation index and duration, parturition, maternal care and earlypostnatalpup development consisting of mortality, clinical signs, body weight and macroscopy).

 

Short description of key information:
No adverse effects on development and fertility were noted in the gavage screening study in rats at dose levels of 100, 300 and 1000 mg/kg bw (OECD 422, GLP).

Effects on developmental toxicity

Description of key information

No adverse effects on development and fertility were noted in the gavage screening study in rats at dose levels of 100, 300 and 1000 mg/kg bw (OECD 422, GLP).

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

Quality of whole database:

Screening study only. Teratogenicity not examined.

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

Dangerous Substance Directive (67/548/EEC)

The available screening study is considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for developmental toxicity or fertility under Directive 67/548/EEC, as amended for the 31st time in Directive2009/2/EG. 

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available screening study gives some information for classification purposes under Regulation 1272/2008. No information on teratogenicity is available. As a result the substance is not considered to be classified for developmental toxicity or fertility under Regulation (EC) No. 1272/2008, as amended for the fifth time in Directive EC944/2013.

During the four days covered in the screening study, no effects via lactation were observed.