Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011 - 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well documented, according to OECD guideline and GLP

Data source

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

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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
444-860-9
EC Name:
-
Cas Number:
474510-57-1
Molecular formula:
C21 H24 O4
IUPAC Name:
2-hydroxy-1-(4-(4-(2-hydroxy-2-methylpropionyl) benzyl) phenyl)-2-methylpropan-1-one

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Breeder: Harlan Netherlands
- Age (at start of treatment): 11 weeks
- Acclimatization: 7 days
- Body weight range: 291-371 g (males), 182-213 g (females)
- Diet: ad libitum
- Water: ad libitum
- Fasting: 16h before administration

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12



Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Carboxymethylcellulose sodium salt (0.5%)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
-The dose formulations were prepared weekly
- Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): As used in previous dose range-finding study
Details on mating procedure:
During the 14-day pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The day with positive mating was designated day 0 post coitum.
- M/F ratio per cage: 1:1
- Length of cohabitation: until evidence of copulation was observed
- Proof of pregnancy: the daily vaginal smear was sperm positive, or a copulation plug was observed, referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The samples were analyzed following an analytical procedure provided by the Sponsor and
adapted at Harlan Laboratories. The test item was used as the analytical standard. Analyzed
samples were not discarded without written consent from the study director.
Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Füllinsdorf /
Switzerland. The samples were not discarded without written consent from the study director.
Duration of treatment / exposure:
Males: 4 weeks
Females: approx. 7 weeks
Frequency of treatment:
once daily
Details on study schedule:
First day of dosing: day 1 of pre-pairing
Pre-pairing: 14 days
Pairing: 14 days maximum
Gestation: approx. 21 days
End of treatment: day 4 post partum
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 30, 90 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Doses were selected based upon a previously conducted dose range-finding study in rats
Positive control:
not applicable

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: daily

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of the scheduled necropsy from 5 males from each group, blood samples from 5 lactating females from each group were obtained on day 5 post partum
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, 18h
- How many animals: 5 per sex and group
- Parameters checked in table [1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of the scheduled necropsy from 5 males from each group, blood samples from 5 lactating females from each group were obtained on day 5 post partum
- Animals fasted: Yes, 18h
- How many animals: 5 per sex and group
- Parameters checked in table [2] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: one time during the study
- Dose groups that were examined: 5 animals per sex and group
- Battery of functions tested: cage side observations, hand-held observations, open field observations, reflexes, hind / fore limb strength
Oestrous cyclicity (parental animals):
not determined
Sperm parameters (parental animals):
Spermatogenesis stages investigation and histopathology of interstitial cell structure.
Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no (not necessary)

PARAMETERS EXAMINED
The following parameters were examined in F1offspring: number and sex of pups, stillbirths, live births, presence of gross anomalies, survival rate, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
Dead pups, except those excessively cannibalized, were examined macroscopically.
Postmortem examinations (parental animals):
SACRIFICE
All animals sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table)
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 4 days of age.
- All animals were subjected to postmortem examinations (macroscopic examination)

GROSS NECROPSY
- Gross necropsy consisted of a detailed macroscopic examination
Statistics:
Means and standard deviations of various data were calculated.
The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
fertility indices, mean precoital time, post-implantation losses, mean litter size
Offspring viability indices:
pup sex ratios and viability indices

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

Mortality and General Tolerability
At 90 mg/kg body weight/day, one male was killed for ethical reason on day 9 of treatment in the pre-pairing period and one female died spontaneously on day 2 of pairing period (on day 16 of treatment). After histopathological examination it was concluded that the cause of death of the
female was likely the thrombosis in jejunum and, therefore, this death was considered to be incidental and not test item related. Histopathological examination of the male showed slight diffuse hyperkeratosis in forestomach and minimal erosion in glandular stomach, signs of moderate stress in thymus and adrenal glands as well as slight diffuse atrophy in the prostate, coagulating gland and seminal vesicles. The body weight and food consumption of this male was rapidly decreasing from the second day of treatment onwards probably due to individual sensitivity. Since other males at 90 mg/kg body weight/day, started to show body weight and food consumption reduction later in the treatment period (the body weight started to decrease from the pairing period onwards and the food consumption was decreased in the second week of pre-pairing period), relationship to the treatment could not be excluded.
Test item-related clinical signs were observed of males and females receiving 90 mg/kg body weight/day of Irgacure 127. Hunched posture, ruffled fur and discoloured feces were seen in the pre-pairing and pairing period in males and in the pre-pairing, through pairing and gestation
period in females.
Functional Observational Battery
None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect.
Mean body temperature in males was statistically significantly lower at the dose level of 90 mg/kg body weight/day compared to the control group (37.3 °C compared to 38.1 in the control group) and it was marginally lower when compared to historical control data range (37.5 - 39.1 °C). Additionally, slightly lower locomotor activity of this group was seen, thus a treatment-related effect can not be excluded.
Locomotor Activity
For females at the dose level of 90 mg/kg body weight/day, total level of locomotor activity was statistically significantly reduced. In males the total level of activity was lower when compared to controls or other test item-treated groups but it did not reach statistical significance. The reduced locomotor activity in animals receiving 90 mg/kg body weight/day of Irgacure 127 was considered to be a test item-related effect.
Food Consumption
The mean food consumption of males receiving 90 mg/kg body weight/day of Irgacure 127 was statistically significantly lower on the second week of pre-pairing period, and for females during the whole pre-pairing period. This was considered to be a test item-related effect.
Body Weights
From day 5 of the pre-pairing period the mean body weight of males receiving 90 mg/kg body weight/day of Irgacure 127 was minimally decreased throughout the study when compared to the control group, although not reaching a level of statistical significance. The mean body weight gain of males receiving 90 mg/kg body weight/day of Irgacure 127 was lower from day 5 of pre-pairing period when compared to controls and it was statistically significantly reduced between days 8 and 14. In correlation with the low food consumption this was considered to be a test item-related effect.
Clinical Laboratory Investigations
The assessment of the hematology data did not reveal any test item-related effect in any dose group in any sex. Minimally increased total cholesterol level of females at the dose level of 90 mg/kg body weight/day was observed.
Reproduction and Breeding Data
All pairs mated. Mean precoital time, fertility index and conception rate were not affected by the treatment with the test item. Mean number of corpora lutea and implantations per dam and the post-implantation loss were not affected by the treatment with the test item. The mean postnatal
loss between days 0 and 4 post partum was higher and the mean number of living pup on day 4 post partum were lower consequently the viability index was statistically significantly lower in the high dose group therefore these were considered to be test item-related effects.
Organ Weights
Absolute and relative mean liver weights (both relative to body weight and relative to brain weight) were statistically increased in males and females of group 4 (90 mg/kg body weight/day). Additionally, absolute and relative mean thymus weights were statistically significantly decreased in females of the 90 mg/kg body weight/day group.
Macroscopical Findings and Histopathological Examinations
Treatment-related adverse local irritative effects in the stomach were observed in all test item treated groups at incidences of 1, 2 and 2 animals at the dose levels 10, 30 and 90 mg/kg body weight/day, respectively. The reduced size in thymus in females, occurring in increased incidences, at ≥ 10 mg/kg body weight/day correlated histopathologically with athropy and reduced absolute and relative thymus weights only at the high dose levels. Although this is considered to be most likely to be stress related a test item-relationship cannot be excluded at the 90 mg/kg body weight/day dose level. Treatment-related diffuse liver cell hypertrophy at 90 mg/kg body weight/day was considered to be adaptive. This hypertrophy was associated with follicular cell hypertrophy in thyroid glands in some males and females at 90 mg/kg body weight/day. Most probably the enhanced liver cell
metabolism resulted in an acceleration of thyroid hormone breakdown with consequent thyroid follicular cell hypertrophy. This finding is commonly seen in rats after being exposed to xenobiotics.
No histological evidence of toxicological properties in the reproductive organs and tissues examined were detected at 90 mg/kg body weight/day.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mortality, clinical signs, body weight and food consumption effects, increased liver and thymus weights at 90 mg/kg/d
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
90 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female

Results: F1 generation

Details on results (F1)

Findings at First Litter Check and during Lactation
The mean litter size of dams receiving 90 mg/kg body weight/day of Irgacure 127 was slightly lower when compaired to controls. Mean postnatal loss between days 0 and 4 post partum was higher and the mean number of living pup on day 4 post partum was lower in the group receiving
90 mg/kg body weight/day. However, only 2 litters out of 4 had a postnatal pup loss of higher than one (affected litters: 72, 74, 79, 80). The sex ratio was not affected. No abnormal pup was noted at any dose level.
At the dose level of 90 mg/kg body weight/day, three male pups in three different litters (nos. 72, 74, 79) had no milk in stomach at first litter check and they were missing afterwards on days 4, 2, 2 post partum, respectively.
Pup Weights Up to and Including Day 4 Post Partum
Mean pup weights on day 1 post partum were unaffected, whereas, the mean body weight of pups on day 4 post partum (dose level of 90 mg/kg body weight/day) was significantly lower and therefore considered to be a test item-related effect.
Macroscopical Findings
At necropsy of pups, there were no abnormal findings noted.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: lower pup body weight, decreased viability index

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion