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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 November to 24 December 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study undertaken at a GLP accredited laboratory, to internationally accepted guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
LumiNova Green
IUPAC Name:
LumiNova Green
Details on test material:
- Substance type: Phosphorescent pigment for inks and plastics.
- Physical state: Powder
- Analytical purity: > 99%
- Lot/batch No.: PM-305
- Expiration date of the lot/batch: 21January 2013
- Storage condition of test material: Room temperature (approximately 20°C) in the dark and dessicated
- Other: Date of receipt 14 February 2011

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Commercial laboratory animal supplier
- Age at study initiation: (P) 10 wks;
- Weight at study initiation: (P) Males: 336 - 384 g; Females: 216 - 278 g
- Fasting period before study: No
- Housing: The gridded cages used during pairing were suspended over trays covered with absorbent paper which was changed daily. For cages with solid floors, wood based material was used as bedding and was sterilised by autoclaving and changed at least twice each week.
- Diet: The animals were allowed free access to a standard rodent diet (SDS VRF1 Certified diet).
- Water: Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 40 - 70
- Air changes (per hr): Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod: 12 hrs dark /12 hrs light):

IN-LIFE DATES: From: 02 November 2011 To: 24 December 2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance, LumiNova Green, was prepared for administration as a series of graded concentrations in the vehicle. The required amount of test substance was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogeniser. The remaining concentrations were formulated in ascending order using the same method. As LumiNova Green is an extremely dense test material, vigorous mixing for at least five minutes was required in order to achieve a homogenous suspension prior to sampling or dose administration.

DIET PREPARATION
- Rate of preparation of diet (frequency): The test substance was used as supplied. Formulations were prepared daily for the first five days until stability results indicated that weekly preparation was possible.
- Storage temperature of food: Formulations were stored refrigerated (approximately 4°C) before use.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): cages with solid floors, wood based material was used as bedding and was sterilised by autoclaving and changed at least twice each week.
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A representative sample (either 1 mL, 2 mL or 5 mL, accurately weighed) of test formulation was mixed with hexane to dissolve the olive oil. The sample was then centrifuged (3500 rpm, 1 minute) to separate the test material sediment from the olive oil/hexane supernatant. The supernatant was then poured through the pre weighed filter paper (Millipore fluropore 0.45μm) using a bottle top filter apparatus. The filter paper was allowed to dry (Ca 5-10 minutes, ambient) and re-weighed to determine the mass of the sample.
Duration of treatment / exposure:
The adults were treated daily for a minimum of 15 days before pairing until Day 6 after the birth of the F1 generation. A similarly constituted Control group received the vehicle, olive oil, at the same volume-dose throughout the same period.
Frequency of treatment:
Daily
Details on study schedule:
- Age at mating of the mated animals in the study: 11 - 13 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg / kg bw / day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
300 mg / kg bw / day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1000 mg / kg bw / day
Basis:
nominal conc.
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses used in this study (0, 100, 300 and 1000 mg/kg/day) were selected in conjunction with the Sponsor with reference to a previous toxicity study. In that study, daily administration at dose levels up to 1000 mg/kg/day produced minimal toxicity. Based on this data, a high dose level of 1000 mg/kg/day (the limit dose) was selected. Low and intermediate dose levels of 100 and 300 mg/kg/day were selected to give an approximate three-fold difference between dose levels to establish the NOEL and to investigate any possible dose response.
- Rationale for animal assignment (if not random):
- Other:
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule: days 0, 7, 14 and 20 after mating and days 1 and 7 of lactation to monitor general health

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded during acclimatisation, on the day that treatment commenced (Week 0), weekly thereafter and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

OTHER:
Oestrous cyclicity (parental animals):
For 15 days before pairing, daily vaginal smears were taken from all females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed.
Sperm parameters (parental animals):
No data
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 determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: F0 males were killed after Day 7 of lactation of the females.
- Maternal animals: F0 females were killed on Day 7 of lactation.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated were prepared for microscopic examination and weighed, respectively.

Epididymides (L&R)
Testes (L&R)
Pituitary
Prostate
Seminal vesicles with coagulating glands
Postmortem examinations (offspring):
GROSS NECROPSY
For offspring surviving to scheduled termination, a careful external examination was performed for gross abnormalities and externally normal offspring were discarded without internal examination. Externally abnormal offspring were internally examined and any abnormal tissues were retained in an appropriate fixative. Additionally the following procedures were applicable:
Premature deaths: Missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before Day 7 of age were examined as detailed above. The necropsy also included an assessment for the presence of milk in the stomach, where this was possible.
The retained tissues were checked before disposal of the carcass.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated were prepared for microscopic examination and weighed, respectively.

Epididymides (L&R)
Testes (L&R)
Pituitary
Prostate
Seminal vesicles with coagulating glands
Offspring viability indices:
Control 100%
100 mg/kg 100%
300 mg/kg 100%
1000 mg/kg 98.6%

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS) There were no deaths, and the general appearance and behaviour of the animals were not affected by treatment. Clinical signs were limited mainly to hairloss from the forelimbs during lactation. This was seen in all groups and is considered not related to treatment with LumiNova Green

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS) There was no clear effect of treatment on the gains of males receiving LumiNova Green at 100, 300 or 1000 mg/kg/day between Weeks 1 and 4 of treatment. The group mean bodyweight gain of males receiving LumiNova Green at 100, 300 and 1000 mg/kg/day during the last two weeks of treatment was slightly lower than that of the Controls.
Individual values were highly variable, and in the absence of any other finding, the significance of this slightly low bodyweight gain is unclear.
There was no effect of treatment on bodyweight gains of females receiving LumiNova Green at 100 or 300 mg/kg/day in Weeks 1 and 2 of treatment and throughout gestation. Bodyweight gain of females receiving 1000 mg/kg/day was slightly higher than Controls during Weeks 1 and 2, but similar to that of the Controls throughout gestation and there were no intergroup differences in bodyweight/gains during Days 1-7 of lactation

There was no clear effect on food consumption for males or females in Weeks 1 and 2 before pairing, or for females throughout gestation and Days 1-7 of lactation.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS) Oestrous cycle length was unaffected by treatment

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS) Mating performance and fertility as assessed by percentage mating, conception rate and fertility index, were unaffected by treatment. One pairing (M40xF80) in the 1000 mg/kg/day group was infertile, and could probably be attributed to the male (M40) which had small reproductive organs. The isolated nature of this finding suggests that it was unrelated to
treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS) Organ weights were not affected by treatment. The low group mean values for testis and epididymal weight for the high dose males reflects abnormally low values for the infertile male and do not suggest any treatment related effect

GROSS PATHOLOGY (PARENTAL ANIMALS) There were no findings at macroscopic examination that could be attributed to treatment with
LumiNova Green.

HISTOPATHOLOGY (PARENTAL ANIMALS) Animals killed after treatment period;
No test article related changes were observed in the testes, epididymis and ovaries of animals given LumiNova Green up to 1000 mg/kg/day
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.
Incidental findings
All other histological changes were considered to be unrelated to treatment including bilateral absence of spermatozoa in the epididymis and severe bilateral atrophy of the seminiferous tubules and bilateral slight interstitial cell hyperplasia observed in the testes of animal no. 40 (High dose male). These findings in the testes and epididymis correlated with small size of these organs observed during macroscopic examination.

OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no effects observed

Results: F1 generation

General toxicity (F1)

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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING) No data

CLINICAL SIGNS (OFFSPRING) No data

BODY WEIGHT (OFFSPRING) No data

SEXUAL MATURATION (OFFSPRING) No data

ORGAN WEIGHTS (OFFSPRING) No data

GROSS PATHOLOGY (OFFSPRING) Macroscopic examination of offspring which died or were killed for welfare reasons generally confirmed the last in-life finding of no milk present in stomach. No other macroscopic abnormalities were found amongst these animals. Macroscopic examination of offspring killed at scheduled termination on Day 7 of age revealed no abnormality

HISTOPATHOLOGY (OFFSPRING) No data

OTHER FINDINGS (OFFSPRING) No data

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no effects observed

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
It is concluded the No-Observed-Adverse-Effect-Level (NOAEL) for LumiNova Green (a phosphorescent pigment for inks and plastics) was 1000 mg/kg/day (the limit dose) for reproductive performance and development in the CD rat following oral gavage administration in a standard OECD 421 screening test.

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